Ability Of Hematopoietic Stem Cells Research Articles

FoxO1 regulates human haematopoietic stem cells self-renewal and engraftment.

Hematopoietic stem cell transplantation (HSCT) is one of the most effective ways to treat hematological malignant diseases, but the traditional culture of hematopoietic stem cells (HSCs) in vitro will soon lose their ability to self-renewal or differentiate into multilineage blood cells. To determine whether Forkhead boxO1 (FoxO1) is implicated in the development of HSCs, lentiviral vectors expressing knockdown (KD) or overexpression (OE) of FoxO1 were utilized in fetal liver-derived hematopoietic stem and progenitor cells (FL-HSPCs). The impacts on the proliferation and hematopoietic differentiation of FL-HSPCs were subsequently evaluated via flow cytometry (FCM). Furthermore, the effect of FoxO1-OE on the self-renewal of cord blood-derived hematopoietic stem and progenitor cells (CB-HSPCs) was investigated. Additionally, the transplantation ability of hematopoietic stem cells derived from these CB-HSPCs in mice after secondary transplantation was also assessed by FCM. After knocking down FoxO1 in FL-HSPCs, the apoptosis rate was significantly increased, and the expression of hCD45 was significantly decreased. Conversely, overexpression of FoxO1 reversed this phenomenon, effectively promoting the expansion and differentiation of FL-HSPCs in vitro. Similarly, it was found that FoxO1-OE could effectively enhance the expansion of CB-HSPCs. Furthermore, upon transplantation of CB-HSPCs overexpressing FoxO1 into NSG mice, multilineage human hematopoietic reconstruction was promoted. Notably, the results of secondary transplantation revealed that only the FoxO1-OE group exhibited multilineage reconstitution. In conclusion, our study confirmed that FoxO1-OE could enhance the self-renewal and engraftment of CB-HSPCs.

Role of Intermittent Fasting In Starving Cancer Cells

Introduction. An absolute burden of over 35 million new cancer cases is predicted by 2050. Although significant progress has been made in the field of oncology via the use of surgical removal, radiation treatment, chemotherapy, and the introduction of immunotherapy, the overall rates of survival and prognoses for cancer patients are still unsatisfactory. In the last decade, intermittent fasting (IF) has become increasingly popular for weight control and potential health benefits. Additionally, research has primarily focused on investigating the impact of IF on metabolism, mitochondrial function, stress responses, repair mechanisms, and autophagy. The aim of the study. To make a literature review about the role of intermittent fasting in starving cancer cells. Materials and methods. This narrative review involved a comprehensive search through databases such as PubMed and Google Scholar. Distinct keywords such as - ″intermittent fasting″, ″intermittent fasting regimens″, ″calorie restriction″, ″oncology″, ″chemotherapy″ and ″tumor microenvironment″ were used. Results. IF exhibits significant impacts on the immune system's ability to fight against tumors by strengthening the ability of hematopoietic stem cells to replenish themselves and enhance immunosuppression. Under an IF program, certain tissues and organs exhibit enhanced resilience to various stressors. Emerging research shows that IF has the potential to enhance the effectiveness and tolerance of anticancer medicines, regulate carcinogenic influences, reprogram clock genes' rhythmic expression in tumor environments, inhibit tumor growth by modifying natural processes like insulin signalling, heme oxygenase-1, prevent the proliferation of myeloid-derived suppressor cells and priming the tumor microenvironment to support drug delivery that targets tumors. Cancer treatment via IF notably shields normal cells while raising the efficacy of chemotherapy (CT) and reducing CT-induced inflammation via several immunological, biochemical, and molecular mechanisms. Conclusions. Regularly practising fasting for more than one day may confer notable health benefits by protecting healthy normal cells against the deleterious effects of chemotherapy and radiation. The synergistic therapeutic impact of intermittent fasting alongside chemotherapy on tumors suggests that it enhances the efficacy of chemotherapy while also notably reducing chemotherapy-induced inflammation. While intermittent fasting shows promise for certain cancers, such as breast cancer, its efficacy for other types remains uncertain, necessitating further research and personalized treatment plans.

MODL-10. SEVERELY IMMUNODEFICIENT NOG-EXL MICE ALLOW FOR HUMANIZATION AND DEVELOPMENT OF A HUMAN GLIOBLASTOMA-DERIVED TUMOR MICROENVIRONMENT

Abstract Cellular immunotherapy has resulted in tremendous therapeutic advances across liquid tumors. However, for solid tumors, including glioblastoma (GBM), significant limitations still exist in successfully translating immune-based adoptive cell therapeutics from the bench to the clinic. The majority of preclinical in vivo GBM models either use human cell lines grown in immunodeficient animals or syngeneic tumors grown in immunocompetent animals. Both approaches lack critical immune components of the tumor microenvironment (TME), thus inherently limiting the ability to adequately model immunotherapies. Here, we demonstrate the ability of hematopoietic stem cells (HSCs) from a GBM patient to successfully engraft in the severely immunodeficient NOG-EXL mouse strain and generate a patient-derived immune system. Intraosseous injections of a low number (10,000) of CD34+ HSCs, obtained from a bone marrow draw from the patient at the time of tumor resection, resulted in an average of 20% human CD45+ cells in peripheral blood 17 weeks post-transplant. GBM organoids, established from the same patient, were implanted intracranially at Week 18 and allowed to grow for 5 weeks without significant clinical evidence of graft-vs-host disease or macrophage activation syndrome. Immunohistochemical and flow cytometry analyses of mouse brains, with and without tumors, revealed infiltration of human-derived, CD45+/CD33+ cells, in proximity to the tumor engraftment site. These results demonstrate the ability of patient-obtained CD34+ HSCs to engraft and form a TME in severely immunodeficient mice. Subsequent work will focus on the differences between autologous and allogeneic humanized tumor-bearing mice in the context of the originating tumor. Additional future work will focus on testing cellular therapies directed towards the relevant areas of the autologous model.

The Impact of Prolonged and Intermittent Fasting on PGC-1α, Oct-4, and CK-19 Liver Gene Expression.

Liver stemness refers to the high regenerative capacity of the organ. This intrinsic regeneration capacity allows the restoration of post-resection liver function in up to 50% of liver donors. Liver cirrhosis is one of the terminal liver diseases with a defect in the intrinsic regeneration capacity. Several attempts to restore intrinsic regeneration capacity by conducting in vivo studies on stem cells in various organs have shown the positive impact of fasting on stemness. An increased capacity for stem cell proliferation and regeneration was reported due to fasting. Prolonged fasting (PF) has been reported to maintain the long-term proliferative ability of hematopoietic stem cells. However, clinical trials on intermittent fasting (IF) have not conclusively given positive results for fasting individuals. This research aims to investigate the effect of fasting on liver stemness by comparing the expression of octamer-binding transcription factor 4 (Oct-4), cytokeratin 19 (CK-19), and peroxisome proliferator-activated receptor γ co-activator α (PGC-1α) in liver cells of fasted rabbits with rabbits fed ad libitum. This study compares two types of fasting, which are intermittent (16 hours) and prolonged (40 hours) fasting, for liver stemness and intrinsic regenerative capacity. A total of 18 rabbits were conditioned into 3 different groups. The first group was subjected to an ad libitum diet, the second to intermittent fasting (16-hour fasting), and the third to prolonged fasting (40-hour fasting). Afterward, the RNA was extracted from the liver tissues of each rabbit and analyzed via real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Relative expression was calculated using the Livak method. Compared to the ad libitum diet, a greater increase was reported in PGC-1α, upregulated Oct4, and steady CK-19 gene expressions in the livers of intermittent fasting rabbits. Prolonged fasting increased PGC1α, reduced liver stemness, and a statistically insignificant decrease in intrinsic liver regenerative capacity. Intermittent fasting indicates preferable molecular alterations in liver stemness and intrinsic regenerative capacity compared to prolonged fasting.

Gamma-Tocotrienol Modulates Total-Body Irradiation-Induced Hematopoietic Injury in a Nonhuman Primate Model.

Radiation exposure causes acute damage to hematopoietic and immune cells. To date, there are no radioprotectors available to mitigate hematopoietic injury after radiation exposure. Gamma-tocotrienol (GT3) has demonstrated promising radioprotective efficacy in the mouse and nonhuman primate (NHP) models. We determined GT3-mediated hematopoietic recovery in total-body irradiated (TBI) NHPs. Sixteen rhesus macaques divided into two groups received either vehicle or GT3, 24 h prior to TBI. Four animals in each treatment group were exposed to either 4 or 5.8 Gy TBI. Flow cytometry was used to immunophenotype the bone marrow (BM) lymphoid cell populations, while clonogenic ability of hematopoietic stem cells (HSCs) was assessed by colony forming unit (CFU) assays on day 8 prior to irradiation and days 2, 7, 14, and 30 post-irradiation. Both radiation doses showed significant changes in the frequencies of B and T-cell subsets, including the self-renewable capacity of HSCs. Importantly, GT3 accelerated the recovery in CD34+ cells, increased HSC function as shown by improved recovery of CFU-granulocyte macrophages (CFU-GM) and burst-forming units erythroid (B-FUE), and aided the recovery of circulating neutrophils and platelets. These data elucidate the role of GT3 in hematopoietic recovery, which should be explored as a potential medical countermeasure to mitigate radiation-induced injury to the hematopoietic system.

Tumor Necrosis Factor from Leukemic Microenvironment Provides Non-Leukemic Hematopoietic Stem Cells with Megakaryocyte Lineage Bias

Background: Acute myeloid leukemia (AML) is characterized by proliferation of malignant cells and impairment of normal hematopoiesis, leading to the severe bone marrow failure. Although genetic abnormalities and pathogenesis of AML are now investigated in detail, the precise mechanism impairing normal hematopoiesis in AML is not fully elucidated yet. Hematopoietic stem cells (HSC) retain the self-renewal capacity together with multipotent differentiation potential into all mature blood cells, playing a major role in maintaining the normal hematopoiesis. The ability of HSC is maintained by complex network of intrinsic factors and external stimulation from microenvironment called as HSC niche. According to these backgrounds, we hypothesized that the impairment of normal hematopoiesis in AML is caused by abnormal function of non-leukemic (nl) HSC. To verify this hypothesis, we isolated nl-HSC and HSC niche cells from MLL-AF9-induced AML mice and further analyzed their function and gene expression profiles. Results: The frequencies of nl-HSC/hematopoietic progenitor fraction (Lin-cKit+Sca1+ cells, HSPC) were significantly increased at the initiation phase of leukemic proliferation in AML mice (leukemic cells : 10-30% in BM)（normal: 0.76+/-0.34%, AML: 2.07 +/-0.93 %, p<0.001）. Surface expression level of CD41 was also upregulated (normal: 15.1+/- 0.526%, AML: 25.4+/- 9.28%, p<0.05). Interestingly, the results of in vitro culture of nl-HSPC demonstrated that they proliferated more efficiently than HSPC from normal mouse BM, and that they tended to differentiate into megakaryocyte (MgK) lineage. Gene set enrichment analysis revealed that MgK-related gene sets and several inflammatory cytokines signaling pathways, including tumor necrosis factor (TNF) pathway, were statistically enriched in nl-HSC. These data suggested that nl-HSC/HSPC retain the MgK-biased character. On the other hands, the gene expression profiles of perivascular stromal cells (ICAM1+CD31-CD45-TER119-cells) in AML mice demonstrated the significantly decreased expression of HSC-supporting factors (e.g., lpl, ptn, rspo2, angptl4, kitl, cxcl12), when compared with those in normal mice. These data suggested that the changes of multiple external humoral factors from leukemic environment induce functional alternation of nl-HSC. To further investigate the role of inflammatory humoral factors in HSC self-renew and differentiation, we next utilized polyvinyl alcohol (PVA)-based ex vivo HSC expansion system and focused on the role of TNF signaling. In the analysis of highly purified normal HSC (CD150+CD48-CD34-cKit+Sca1+Lin-cells) with PVA, TNF alpha increased both proliferation and MgK differentiation. UMAP analysis of single cell RNA sequence data for ex vivo-expanded HSCs with TNF alpha showed the increased frequencies of the clusters expressing MgK-related genes (e.g., itga2b and gp1ba) and relatively decreased immature HSC related genes (e.g., hlf and procr). The results of competitive repopulation assay using ex-vivo expanded normal HSC with TNF alpha stimulation in HSC promoted myeloid differentiation of HSC, rather than self-renew and expansion of HSC. Conclusion: Our data suggested that TNF alpha from leukemic microenvironment may play a key role for the driver signaling of HSCs to MgK, eventually resulting in the impairment of normal HSC maintenance.

Deficiency of the sedoheptulose kinase (Shpk) does not alter the ability of hematopoietic stem cells to rescue cystinosis in the mouse model

Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin, and leading to multi-organ degeneration including kidney failure. A clinical trial for cystinosis is ongoing to test the safety and efficacy of transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) ex vivo gene-modified to introduce functional CTNS cDNA. Preclinical studies in Ctns−/− mice previously showed that a single HSPC transplantation led to significant tissue cystine decrease and long-term tissue preservation. The main mechanism of action involves the differentiation of the transplanted HSPCs into macrophages within tissues and transfer of cystinosin-bearing lysosomes to the diseased cells via tunneling nanotubes. However, a major concern was that the most common cystinosis-causing mutation in humans is a 57-kb deletion that eliminates not only CTNS but also the adjacent sedopheptulose kinase SHPK/CARKL gene encoding a metabolic enzyme that influences macrophage polarization. Here, we investigated if absence of Shpk could negatively impact the efficiency of transplanted HSPCs to differentiate into macrophages within tissues and then to prevent cystinosis rescue. We generated Shpk knockout mouse models and detected a phenotype consisting of perturbations in the pentose phosphate pathway (PPP), the metabolic shunt regulated by SHPK. Shpk−/− mice also recapitulated the urinary excretion of sedoheptulose and erythritol found in cystinosis patients homozygous for the 57-kb deletion. Transplantation of Shpk−/−-HSPCs into Ctns−/− mice resulted in significant reduction in tissue cystine load and restoration of Ctns expression, as well as improved kidney architecture comparable to WT-HSPC recipients. Altogether, these data demonstrate that absence of SHPK does not alter the ability of HSPCs to rescue cystinosis, and then patients homozygous for the 57-kb deletion should benefit from ex vivo gene therapy and can be enrolled in the ongoing clinical trial. However, because of the limits inherent to animal models, outcomes of this patient population will be carefully compared to the other enrolled subjects.

Bone Marrow Stromal Antigen 2 Facilitates Activation of Hematopoietic Stem Cell through the Induction of ERK Signaling

Chronic infections are prevalent worldwide and contribute to bone marrow failure syndromes. Long-term homeostatic production of all blood cells is dependent on the ability of hematopoietic stem cells (HSCs) to remain mostly quiescent and, upon activation, must carefully balance between their ability to self-renew and differentiate. However, infections trigger inflammatory cytokines, such as interferon-gamma (IFNy), that facilitate HSC proliferative exhaustion by disrupting both quiescence and self-renewal, thereby contributing to cytopenias and bone marrow failure syndromes. Our previously published results indicate that the IFN-induced surface protein, Bone Marrow Stromal Antigen 2 (BST2), facilitates HSC activation, and the loss of BST2 protected against the depletion of HSCs during chronic infection. However, the mechanism by which BST2 impacts HSCs activation is unknown. BST2 is known to promote activation of intracellular signaling in both dendritic cells and cancer cell types. In particular BST2 is a known activator of the NFkB signaling pathway. We hypothesized that BST2 could promote activation of HSCs through the induction of intracellular signaling pathways.Intracellular signaling pathways have been shown to be important for the activation of not only HSCs but also downstream immune cells. Indeed, the loss of BST2 has been shown to impair the function of T cells, NK cells, and antigen presenting cells in the presence of viral infections via impaired induction of NFkB signaling. However, there have been no studies on the role of BST2 during bacterial infections. To evaluate the role of BST2 in hematopoiesis during a bacterial infection, we obtained BST2KO mice and characterized hematopoietic populations in the presence or absence of Mycobacterium avium infection, a model bacterial infection that generates high levels of IFNy. We observed no changes in activation or production of cytokines from macrophage, NK, CD8, or neutrophil populations. Surprisingly we observed enhanced dendritic cell activation at day 15, 30, and 42 as measured by co-stimulatory molecules and MHC II expression. Additionally, we observed increased tetramer-specific T cell numbers early on during the infection. Interestingly, these results pair with decreased bacterial load at day 30 and day 42 despite having the similar spleen sizes. These results suggest that BST2 regulates the activation of dendritic cells, which may trigger more robust earlier T cell responses early in the course of infection.Our previous work showed that the loss of BST2 protects against HSC activation by displacement of HSCs from CXCL12-abundant reticular (CAR) cells in the niche. To determine whether HSC activation is related to distancing from the vascular niche or increased proximity to an activated osteoblastic niche, we performed intravital imaging using osteocalcin-GFP reporter mice. We observed no change in proximity to the osteoblastic niche after administration of recombinant IFNy using either WT or BST2KO HSCs. These results suggest that the effects of BST2 may be more related to distancing from CAR cells than migration towards other niche locations.To evaluate the mechanisms underlying BST2-dependent HSC activation, we performed RNA sequencing on WT versus BST2KO HSCs in the presence of IFNy. GSEA-pathway analysis of differentially expressed genes revealed that BST2KO HSCs have a decreased response to IFNy and diminished activation of the P13K-AKT-MTOR signaling pathways. Western blot analysis on hematopoietic stem cell progenitor cells (HSPCs) indicate a decrease in phosphorylation of the ERK signaling pathway in BST2KO HSPCs. These results were analogous to work performed in 32D cells in vitro, in which ERK-phosphorylation has been shown to be important for both HSC maintenance and proliferation. Future studies are aimed at assessing the impact of ERK inhibition on HSC activation during various inflammatory stresses.Collectively our results show that the loss of BST2 in downstream immune cells surprisingly enhances dendritic cell and T cell activation in response to bacterial infection. BST2 enhances HSC activation through activation of downstream ERK signaling. Future targeting of BST2 and/or ERK signaling could have important consequences for patients that suffer from cytopenias, bone marrow failure, and engraftment failure due to high levels of inflammatory stimuli. DisclosuresNo relevant conflicts of interest to declare.

Effect of Laptm4b Deletion on Hematopoietic Stem and Progenitor Cells Homeostasis in Mice

To investigate the effect of lysosomal-associated protein transmembrane-4 Beta(Laptm4b) deletion on hematopoietic stem/progenitor cells (HSPCs) homeostasis in mice. The hematopoietic system specific Laptm4b-deficient mice were constructed. The number and proportion of HSPCs (LSK, LT, ST, MPP, etc) in Laptm4b-deficient mice were analyzed by flow cytometry. Single SLAM-HSC cell was sorted by flow sorter and cultured in vitro to measure the effect of Laptm4b deletion on the colony forming ability of hematopoietic stem cells (HSCs). The effect of Laptm4b-deficient on the reconstitution ability of HSCs in mice was detected by competitive transplantation experiment of SLAM-HSC cells. Laptm4b deficiency could moderately upregulate the proportion of T cells in the peripheral blood of the mice, but showed no significant effect on the proportion and number of HSPCs. Laptm4b deletion showed no effect on the reconstruction ability of HSCs after competitive transplantation, but it could inhibit the colony formation of HSCs in vitro. LAPTM4B may play a role in HSCs under the proliferation stress. Laptm4b-deficient in mice hematopoietic system showed no significant effect on the HSPCs homeostasis maintenance and reconstruction ability.

Angelica Sinensis Polysaccharide Mediates Sirtl to Inhibit P53/P21 Pathway and Delay Hematopoietic Stem Cell Aging

To study the effect of Angelica sinensis polysaccharide (ASP) on the expression of Sirt1/p53/p21 in radiation-induced aging hematopoietic stem cells (HSCs) of mice, and to explore the possible mechanism of ASP regulating the aging of HSCs. Methods: C57BL/6J mice were randomly divided into control group, model group and ASP group. The model group was irradiated with X-ray 3.0Gy/8F to establish the aging model of HSCs in mice. ASP group was given ASP intragastric administration during irradiation; The control group and the model group were given equal volume of normal saline. HSCs were sorted by immunomagnetic beads, and cell cycle was detected by flow cytometry The aging cells were observed by α-galactosidase staining. The directional differentiation ability of HSCs was observed by mixed colony culture (CFU-Mix). The expression of Sirtl, p53 and p21 protein was detected by Western blot. Results: Compared with the control group. X-ray can significantly increase the proportion of HSCs Gi cells, the rate of SA-α-Gal positive cells and the expression of p53 and p21 protein in aging group (P&lt;0.05).The expression of Sirtl and mixed colony forming ability were decreased (P&lt;0.05). Compared with the model group, ASP inhibited the increase of aging HSCs Gicell ratio, SA-fJ-Gal positive cell rate and p53 and p21 protein expression (P&lt;0.05). At the same time, Sirtl expression and mixed colony forming ability were increased (P&lt;0.05). conclusion: ASP may inhibit p53/p21 pathway and delay the aging of mouse HSCs by regulating Sirtl.

Mouse parabiosis model promotes recovery of lymphocytes in irradiated mice

Purpose Total body irradiation (TBI) -induced hematopoietic system injury is mainly due to the failure of self-renewal and to the differentiation ability of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) after radiation exposure. The mouse parabiosis model is mainly used in the field of aging research to explore whether circulating factors in peripheral blood can improve the functions of aged tissues and organs. In this study, we generated a mouse model to verify whether non-irradiated peripheral circulation can improve the circulatory environment in irradiated mice and ameliorate TBI-induced hematopoietic system injury. Materials and methods Six- to eight-week-old male C57BL/6 mice were adjoined by a surgical operation. Four weeks later, one mouse in the pair was exposed to 8 Gy or 6 Gy X-ray, and B and T cells in the peripheral blood, bone marrow, spleen, mesenteric lymph nodes and thymus were then detected by flow cytometry. Hematopoietic stem/progenitor cells in bone marrow cells and their levels of ROS and apoptosis were also detected in this study. Results The results showed decreased percentages of B and T lymphocytes in the peripheral blood, bone marrow (BM), spleen and mesenteric lymph nodes (MLNs) in the isotype irradiated mice. The proportions of CD4-positive, CD8-positive, and CD4 and CD8 double-negative cells were also increased, while the proportion of CD4 and CD8 double-positive cells in the irradiated thymus was decreased. Thus, all of the above lymphocyte injuries in the parabiosis model were improved to nearly the levels of the control. We further detected radiation-induced HSC and HPC injury; however, the reduced HSC and HPC numbers, ROS levels and apoptosis percentages were not ameliorated in the parabiotic irradiated mice. Conclusions Above all, our results showed that non-irradiated peripheral circulation can promote the recovery of TBI-induced lymphocyte injury, further indicating that the recovery of immune cells may play a very important role in the repair of TBI-induced damage.

Grape seed proanthocyanidin extract ameliorates ionizing radiation-induced hematopoietic stem progenitor cell injury by regulating Foxo1 in mice

Ionizing radiation (IR)-induced excessive reactive oxygen species (ROS) is an important contributor of the injury of hematopoietic system. Grape seed proanthocyanidin extract (GSPE) is a new type of antioxidant, whereas whether it could ameliorate IR-induced hematopoietic injury remains unclear. Here, we show that GSPE treatment improves the survival of irradiated mice and alleviates IR-induced myelosuppression. Meanwhile, the hematopoietic reconstituting ability of hematopoietic stem cells (HSCs) in mice following irradiation exposure is significantly increased after GSPE treatment. Furthermore, GSPE treatment can reduce IR-induced ROS production and relieve DNA damage and apoptosis in hematopoietic stem progenitor cells (HSPCs). Interestingly, we find that a critical antioxidant-associated gene fokhead box transcription factor O1 (Foxo1) is significantly decreased in HSPCs after irradiation. Consistently, hematopoietic specific deletion of Foxo1 increases the radiosensitivity of mice. Further investigations reveal that GSPE treatment specifically upregulates the expression of Foxo1, as well as its target genes superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2) and catalase (CAT). Importantly, Foxo1 deficiency largely abolishes the radioprotection of GSPE on HSPCs. Collectively, our data demonstrate that GSPE plays an important role in ameliorating IR-induced HSPC injury via the Foxo1-mediated pathway. Therefore, GSPE may be used as a promising radioprotective agent.

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.

Effects of Asphyxia on Colony-forming Ability of Hematopoietic Stem Cell of Cord Blood

Background: Asphyxia is a medical situation resulting from the deprivation of oxygen to a newborn lasting long enough during the birth process to cause physical harm, especially to the brain. Human umbilical cord blood (UCB) is a well-established source of hematopoietic stem/progenitor cells (HSPCs) for allogeneic stem cell transplantation. A low level of O2 in neonates with asphyxia during labor can affect proliferation and differentiation of stem cells in cord blood. Methods: The quality and colony-forming ability of hematopoietic stem cells in the cord blood of neonates with severe asphyxia with Apgar score 3-5or need to cardiac pulmonary resuscitation 5 min after delivery were compared with the group with normal Apgar score. Thereafter, hematopoietic stem cells were isolated, and cells were cultured in an enriched media (MethoCult H4435) special for HSPCs for 7 days to assess the growth and colony formation. Results: Based on the results, there was a significant difference in the number of colonies of RBC (P=0.0016) and WBC precursor (P=0.006), in a plate with 104 cord blood hematopoietic stem cells in newborns exposed to hypoxemia during labor. Conclusion: Umbilical cord blood is valuable for its content of stem cells. Severe hypoxia in the perinatal period does not negatively affect the viability of UCB-derived HSPCs to grow and form colonies. Furthermore, it was found that transient severe asphyxia does not exert negative effects on the banking quality of HSPCs for likely problems in the future.

Regulation of Ribosomal RNA Synthesis in Myeloid Progenitors By Cell Type Specific Transcription Factors

Haematopoiesis relies on the ability of hematopoietic stem cells to progress through a systematic hierarchy to produce lineage-restricted progenitors that terminally differentiate into phenotypically distinct types of mature hematopoietic cells. This process is precisely coordinated by the combinatorial activity of lineage-specifying transcription factors (TFs). Indeed, the critical transcriptional program of every hematopoietic cell type, and indeed of all cell types throughout the body, requires a set of core TFs for its proper execution. A frequently-overlooked component of the cellular transcriptional program is the transcription of ribosomal RNA (rRNA), the major component of ribosomes. Ribosomal RNA comprises 90% of total cellular RNA, and its transcription from hundreds of rDNA genes by RNA polymerase I is one of the most intense transcriptional processes in the cell. Different progenitor and mature cell types in the hematopoietic tree have different sizes, ribosome abundances, and rates of protein synthesis. Tight control of ribosome abundance is essential for a normal cellular proteome, and different cell types within the hematopoietic tree have varied rRNA transcription rates. However, there has been limited study of the molecular basis of lineage-specific regulation of rDNA transcription in hematopoiesis. We explored the binding to rDNA of over twenty key hematopoietic TFs that were determined by the Broad Institute DepMap database to be crucial for survival of hematopoietic cell lines. Using a customized bioinformatics pipeline, we mapped over three hundred ChIP-Seq datasets for these factors (generated by us as well as publicly available from ENCODE and GEO) to mouse and human rDNA assemblies, and found that several essential hematopoietic TFs such as MYC, MYB, RUNX1, PU.1, CEBPA and others bind to rDNA at conserved sites and motifs (Fig A). MYC is well-known as a master regulator of rDNA, and RUNX1 was recently reported to bind rDNA, but most of the others have never been linked to rDNA, and their functional roles in regulating rDNA transcription have not been explored. We picked for further study CEBPA, a crucial TF required for specification of granulocyte-monocyte progenitors (GMPs). For our experiments, we used the mouse HOXA9-ER cell line, which mimics GMPs. We used CRISPR/Cas9 and homologous recombination to fuse FKBPV degron into bi-allelic endogenous loci of the Cebpa gene in HOXA9-ER cells, and, upon addition of dTAG-13 (the ligand for FKBPV), the CEBPA-FKBPV fusion protein could be rapidly degraded within 2 hours (Fig B, C), providing us an experimental system to study the immediate consequences of CEBPA loss. In order to quantify the rate of rRNA transcription, we devised an assay titled "47S FISH-Flow" that combined fluorescent in-situ hybridization (FISH) using probes against nascent 47S rRNA with flow cytometry (Fig D, E). This assay not only allows us to quantify the rate of rRNA transcription on a per-cell basis in millions of cells, but also allows us to separately gate and quantify rRNA transcription in different stages of the cell cycle, eliminating a major confounder in bulk cell studies - cell cycle distribution. Using 47S FISH-Flow, we observed that degradation of CEBPA in the HOXA9-ER mouse GMP cell line led to decrease in synthesis of 47S rRNA within hours (Fig F) before any change in cell cycle or growth kinetics, and was followed by growth arrest in 24 hours. In summary, we show that several critical hematopoietic TFs show abundant, conserved binding to rDNA, and the depletion of CEBPA rapidly reduces nascent rRNA, indicating that it directly promotes rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by a wide range of lineage-specific hematopoietic TFs. Figure Disclosures No relevant conflicts of interest to declare.

Regimen-dependent synergism and antagonism of treprostinil and vildagliptin in hematopoietic cell transplantation

The cell dose in umbilical cord blood units is a major determinant for the outcome of hematopoietic cell transplantation. Prostaglandin analogs and dipeptidylpeptidase-4 (DPP4/CD26)-inhibitors enhance the ability of hematopoietic stem cells (HSCs) to reconstitute hematopoiesis. Here we explored the synergism between treprostinil, a stable prostaglandin agonist, and the DPP4/CD26-inhibitor vildagliptin. The combination of treprostinil and forskolin caused a modest but statistically significant increase in the surface levels of DPP4/CD26 on hematopoietic stem and progenitor cells (HSPCs) derived from murine bone and human cord blood. Their migration towards stromal cell-derived factor-1 (SDF-1/CXCL12) was enhanced, if they were pretreated with treprostinil and forskolin, and further augmented by vildagliptin. Administration of vildagliptin rescued 25% of lethally irradiated recipient mice injected with a limiting number of untreated HSPCs, but 90 to 100% of recipients injected with HSPCs preincubated with treprostinil and forskolin. The efficacy of vildagliptin surpassed that of treprostinil (60% rescue). Surprisingly, concomitant administration of vildagliptin and treprostinil resulted in poor survival of recipients indicating mutual antagonism, which was recapitulated when homing of and colony formation by HSPCs were assessed. These observations of regimen-dependent synergism and antagonism of treprostinil and vildagliptin are of translational relevance for the design of clinical trials.Key messagesPretreatment with treprostinil increases surface levels of DPP4/CD26 in HSPCs.Vildagliptin enhances in vitro migration of pretreated HSPCs.Vildagliptin enhances in vivo homing and engraftment of pretreated HSPCs.Unexpected mutual antagonism in vivo by concomitant administration of vildagliptin and treprostinil.

Role of Rictor in Hematopoietic Stem Cells during Fetal Liver Hematopoiesis

To investigate the effect of Rictor on the hematopoiesis of fetal liver by specific knock-out of Rictor in hematopoietic cells of Vav-Cre mice. E12.5 0.08ee fetal liver cells from the experimental group Vav-Cre; Rictorf/f embryos and control group Rictor f/+ or Rictorf/f embryos were transplanted to recipients respectively to observe the effect of Rictor on reconstitution ability of hematopoietic stem cells. In the meantime, E14.5 0, 10, 20, 40, 60, 80 sorted hematopoietic stem cells from the Vav-Cre; Rictorf/f fetal liver of experimental group and Rictorf/+ or Rictorf/f fetal liver of control group were transplanted in to recipients to analyze the numbers of functional hematopoietic stem cells after Rictor was knocked-out. Furthermore, the self-renewal capacity was investigated by secondary transplantation of BM cells from primary recipients that had been successfully repopulated with E12.5 fetal liver-derived cells and by cell cycle analysis. All the recipients receiving E12.5 Rictorf/+ or Rictorf/f cells were repopulated (8/8, from 2 independent experiments) with an average chimerism of 77.2%±11.1% at 4 months post-transplantation, which resulted in 57 LT-RU per FL. In comparison, 8 out of 8 recipients receiving Vav-Cre; Rictorf/f cells were repopulated with significantly reduced chimerism (37.0%±16.3%) (P＜0.01), which was equivalent to 8 LT-RU per FL. The limiting dilution transplantation experiment showed that there was one functional hematopoietic stem cell out of 17 sorted SLAM cells in the control group, and one functional hematopoietic stem cell out of 39 sorted SLAM cells in the experimental group. The secondary transplantation experiments showed that 2 out of 4 recipients were reconstructed in the control group after 1 month, and 0 was reconstructed in the experimental group by transplanting 4×105 donor cells respectively. What＇s more, the percentage of S/G2/M cells in the experimental group increased when compared with controls. In the process of fetal liver hematopoiesis, the specifically knocking-out the Rictor in hematopoietic system can lead to defect of reconstitution ability, decrease of the functional hematopoietic stem cell numbers and reduction of self-renewal ability of hematopoietic stem cells.

The autophagy Protein Atg5 Plays a Crucial Role in the Maintenance and Reconstitution Ability of Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) in bone marrow are pluripotent cells that can constitute the hematopoiesis system through self-renewal and differentiation into immune cells and red blood cells. To ensure a competent hematopoietic system for life, the maintenance of HSCs is tightly regulated. Although autophagy, a self-degradation pathway for cell homeostasis, is essential for hematopoiesis, the role of autophagy key protein Atg5 in HSCs has not been thoroughly investigated. In this study, we found that Atg5 deficiency in hematopoietic cells causes survival defects, resulting in severe lymphopenia and anemia in mice. In addition, the absolute numbers of HSCs and multiple-lineage progenitor cells were significantly decreased, and abnormal erythroid development resulted in reduced erythrocytes in blood of Vav_Atg5−/− mice. The proliferation of Lin−Sca-1+c-Kit+ HSCs was aberrant in bone marrow of Vav_Atg5−/− mice, and mature progenitors and terminally differentiated cells were also significantly altered. Furthermore, the reconstitution ability of HSCs in bone marrow chimeric mice was significantly decreased in the presence of Atg5 deficiency in HSCs. Mechanistically, impairment of autophagy-mediated clearance of damaged mitochondria was the underlying cause of the HSC functional defects. Taken together, these results define the crucial role of Atg5 in the maintenance and the reconstitution ability of HSCs.

Abstract 1913: Investigation of targeting SKP2 to improve the treatment response in glioma

Abstract Glioma is the most frequent tumor in central nervous system (CNS), constituting about 30% of CNS tumors and 80% of malignant brain tumors. The current standard strategy for glioma treatment is surgery plus adjuvant chemotherapy and concurrent radiotherapy. However, the prognosis of glioma is still poor. It has been proved that the existence of glioma stem cells and the timely repair of DNA damages are the main causes of treatment failure. Skp2 (S-phase kinase associated protein-2), a member of F-box proteins, forms the Skp2 SCF complex with Skp1, Cullin-1, and Rbx1, serves as one of the important E3-ligases in the process of ubiquitination, and is responsible for substrate recognition. Skp2 is overexpressed in a variety of human cancers and promotes cancer progression by inducing p27 degradation. Importantly, Skp2 deficiency profoundly restricts cancer progression in multiple genetic mouse tumor models. Our group has demonstrated that Skp2 is involved in the regulation of pool size and self-renewal ability of hematopoietic stem cells, as well as breast cancer stem cells and nasopharyngeal carcinoma stem cell-like cells. However, the exact role of Skp2 in the regulation of glioma stem cells and DNA damage repair in glioma has not been elucidated yet. We proposed that targeting Skp2 could promote radio- and chemotherapy sensitivity through suppressing function of glioma stem cells. We first detected the expression of SKP2 in glioma tumor tissues and found that SKP2 was highly expressed in glioma and related to the prognosis of glioma patients. We also adopted gene knockdown methods and inhibitor pretreatment with small molecules in order to suppress the level of Skp2, and then checked the sphere formation and self-renewal ability of glioma stem cell upon radio/chemo treatments. In this way we evaluated the role of Skp2 in repressing the glioma cell proliferation and subsequently triggering apoptosis. Our project will clarify the novel mechanisms about how Skp2 is involved in the glioma radio- and chemotherapy resistance, which will provide a potential target to improve the radio/chemo therapeutic effect through targeting one single target. Citation Format: Jing Wang, Hong-Kai Su, Zhong-Ping Chen, Yue Qu, Hai-Ping Cai. Investigation of targeting SKP2 to improve the treatment response in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1913.

Enhanced expressions of FHL2 and iASPP predict poor prognosis in acute myeloid leukemia.

iASPP is a negative regulator of the apoptotic function of p53, and it can enhance the ability of hematopoietic stem cells to self-renew and resist chemo- and radiation therapy. Recent study showed that iASPP could impact the proliferation and apoptosis of leukemia cells by interacting with FHL2. However, whether they have prognostic significance in acute myeloid leukemia (AML) is unknown. Eighty-four AML patients with FHL2 and iASPP expression data from The Cancer Genome Atlas database were enrolled in the study. Patients with high expressions of FHL2 and iASPP had significantly shorter event-free survival (EFS) and overall survival (OS) than patients with low expressions (P = 0.005, P = 0.003, respectively). Univariate analysis indicated that high expressions of FHL2 or iASPP were unfavorable for EFS and OS (all P < 0.05), while multivariate analysis confirmed that high FHL2 expression was an independent risk factor for EFS and OS (all P < 0.05). In patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), however, EFS and OS were not significantly different between FHL2 or iASPP high- and low-expression groups. Our results suggested that high expressions of FHL2 and iASPP were poor prognostic factors for AML, but the prognostic effect might be overcome by allo-HSCT.