Hematopoietic Stem Cells Count Research Articles

CD34+ Hematopoietic Stem Cell Count as a Novel Predictor of Vascular Event in Sickle Cell Disease Patients

Abstract Background Sickle cell disease (SCD) is one of the most common genetic disorders, it not only has hematological pathophysiological consequences, but also has systemic pathological manifestations such as vascular events (VEs). Prior studies have described that VEs are associated with poor outcomes in the short term and decreased quality of life in patients over the long term. There is no standardized method for predicting vascular injury in Sickle cell disease patients. Aim of the Work Our aim is to evaluate circulating CD34 as a novel predictive marker of occurrence of vascular event in Children with Sickle Cell Disease in their steady state and relate levels to severity of VEs in the following 6 months. Patient and Methods This study was an exploratory study, conducted at Pediatrics Hematology- Oncology Unit at Ain Shams University Children’s Hospital, where 50 children with sickle cell disease were enrolled at their steady state (defined as 2 months free from any Vascular Event prior to the study), aged 6 months - 16 years. Flow cytometry was done for quantification of circulating CD34+ at inclusion and compared to age and sex matched control and patients were followed up for 6 months for vascular events. Results There is statistically significant higher level of CD34+ among SCD patients compared to age and sex matched control with p value < 0.001.Circulating CD34+ showed significantly higher level in patients who developed systemic vascular events after 6 months of follow up with p value 0.022. There was no statistically significant correlation between circulating CD34+ and severity of peripheral vascular events (or any need for hospitalization) on follow up. Conclusion CD34+ cell count at steady state may be a promising biomarker of further vascular event in children with SCD.

In vivo and in vitro effects of cord blood hematopoietic stem and progenitor cell (HSPC) expansion using valproic acid and/or nicotinamide

BackgroundHigh self-renewal capacity and most permissive nature of umbilical cord blood (CB) results with successful transplant outcomes but low hematopoietic stem and progenitor cell (HSPC) counts limits wider use. In order to overcome this problem ex vivo expansion with small molecules such as Valproic acid (VPA) or Nicotinamide (NAM) have been shown to be effective. To the best of our knowledge, the combinatory effects of VPA and NAM on HSPC expansion has not been studied earlier. The aim of this study was to analyze ex vivo and in vivo efficacy of VPA and NAM either alone or in combination in terms of expansion and engraftment. MethodsA total of 44 CB units were included in this study. To determine the ex vivo and in vivo efficacy, human CB CD34+ cells were expanded with VPA and/or NAM and colony forming unit (CFU) assay was performed on expanded HSPC. Xenotransplantation was performed simultaneously by intravenous injection of expanded HSPC to NOD-SCID gamma (NSG) mice (n = 22). Significance of the difference between the expansion groups or xenotransplantation models was analyzed using t-test, Mann-Whitney, ANOVA or Kruskal-Wallis tests as appropriate considering the normality of distributions and the number of groups analyzed. ResultsIn vitro CD34+ HSPC expansion fold relative to cytokines-only was significantly higher with VPA compared to NAM [2.23 (1.07–5.59) vs 1.48 (1.00–4.40); p < 0.05]. Synergistic effect of VPA+NAM has achieved a maximum relative expansion fold at 21 days (D21) of incubation [2.95 (1.00–11.94)]. There was no significant difference between VPA and VPA+NAM D21 (p = 0.44). Fold number of colony-forming unit granulocyte-macrophage (CFU-GM) colonies relative to the cytokine-only group was in favor of NAM compared to VPA [1.87 (1.00–3.59) vs 1.00 (1.00–1.81); p < 0.01]. VPA+NAM D21 [1.62 (1.00–2.77)] was also superior against VPA (p < 0.05). There was no significant difference between NAM and VPA+NAM D21. Following human CB34+ CB transplantation (CBT) in the mouse model, fastest in vivo leukocyte recovery was observed with VPA+NAM expanded cells (6 ± 2 days) and the highest levels of human CD45 chimerism was detectable with VPA-expanded CBT (VPA: 5.42 % at day 28; NAM: 2.45 % at day 31; VPA+NAM 1.8 % at day 31). ConclusionOur study results suggest using VPA alone, rather than in combination with NAM or NAM alone, to achieve better and faster expansion and engraftment of CB HSPC.

Royal Jelly Increases Hematopoietic Stem Cells in Peripheral Blood: A Double-Blind, Placebo-Controlled, Randomized Trial in Healthy Subjects.

Royal jelly (RJ), produced by honeybees, influences stem cell functions, such as pluripotency maintenance of mouse embryonic stem cells and prevention of aging-related muscle stem cell functional deterioration. Thus, we hypothesized that RJ administration has various health-promoting effects based on stem cells. However, its effects are unknown in humans. In this study, we have attempted for the first time to clarify whether the administration of RJ in humans affects stem cells. This randomized, double-blind, placebo-controlled study was performed on healthy subjects (n = 90) who received protease-treated RJ at a dose of 1200 mg/day or placebo daily for four weeks. Also, the participants with a low number of hematopoietic stem cells (HSCs) in peripheral blood were preferentially selected. HSC counts, endothelial progenitor cell (EPC) counts, blood cell counts in peripheral blood, cytokines in serum, and physical conditions were evaluated. Results and Conclusion. Eligible data from 86 subjects (placebo: 42, RJ: 44) who completed the study were analyzed. There were no significant differences between the two groups regarding the changes in peripheral HSC count (p=0.103), while diastolic blood pressure showed a significant improvement in the RJ group compared to that in the placebo group (p=0.032). The subgroup analysis excluded 14 subjects who complained of cold symptoms at baseline or within five days of the four-week study. The changes in the HSC populations were significantly higher in the RJ group than those in the placebo group (p=0.042). No adverse effects were observed in any of the groups. These results suggest that RJ administration affected the peripheral HSC count and may influence stem cell functions. Further research is needed to reveal the various health-promoting benefits of RJ based on stem cells.

Abstract 20 A Rapid and Sensitive IL-3-Based Assay Reveals How Various Pre-Processing Conditions Affect the Potency of Hematopoietic Stem Cells in Fresh Cord Blood Units

IntroductionOptimized procedures to collect, process, and store cord blood (CB) units are crucial to ensure stem cell product quality; however, current methods to assess hematopoietic stem cell (HSC) potency are time-consuming or lack sensitivity. Therefore, developing methods that rapidly and accurately portray HSC potency in fresh blood is important to help optimize clinical outcomes.ObjectiveWe sought to adapt to fresh CB a potency assay previously developed for HSCs contained in cryopreserved CB [Simard et al. Transfusion. 2019;59(6):2074-2083.]. We next used the assay to evaluate the impact of various CB processing conditions on HSC potency.MethodsFresh CB units were exposed to various pre-processing storage conditions (ie, temperature changes and various anticoagulants). Units were then analyzed at various time points up to 96 hours using traditional iShage and colony-forming assays, in parallel with the IL-3-based potency assay.ResultsFor CB units maintained at room temperature (RT), the potency measured with the IL-3-based assay (ie, the proportion of IL-3-responsive CD34+ cells as measured by pStat5 activation) linearly decreased over time. A potency of 69% was observed when tested within 0-24 hours, compared with 56% and 40% in the 24-48–hour and the 48-72–hour groups, respectively. In addition, the IL-3-based assay showed that pre-processing storage at 4°C prevented this loss of HSC potency. By contrast, the HSC count by iShage and the CFU numbers remained unchanged with time or temperature variations.DiscussionThe newly developed IL3-based assay appears reliable and sensitive to characterize the potency of CD34+ cells in fresh CB. In addition, this assay indicated that HSC potency linearly decreased with time when CB units were maintained at RT but remained stable when units were stored at 4°C pre-processing. This temperature-related difference was not observed with traditional assays, indicating that the test could be a complementary tool to characterize CB units in research and operational settings.

The proteome signature of cord blood plasma with high hematopoietic stem and progenitor cell count

Hematopoietic stem and progenitor cells (HSPC) from umbilical cord blood (UCB) are used for transplantation to treat blood disorders. Methods to estimate the HSPC count in umbilical cord blood, and thereby identify high-value blood units, are time-consuming and costly. Recent studies indicate that the UCB plasma protein composition relates to the HSPC count. We compared the plasma proteome of UCB with high vs low HSPC cell count (>115 × 106 vs < 51 × 106 CD34+ cells l−1) by using a combination of global untargeted MS quantitative proteomics and targeted proximity extension assay (PEA) proteomics. For the MS platform, 96 proteins differed significantly between the CD34+ groups, and out of these, 44 proteins showed more than a two-fold difference. Seven pathways were enriched in high CD34+ samples, including pathways relating to platelets, coagulation, and lipid transport. For the PEA platform, 61 proteins were differentially abundant, and among these 7 proteins showed more than a two-fold difference between groups. In the PEA data, a high CD34+ cell count was associated with a protein hub with functions in platelet degranulation. We conclude that the HSPC count is related to the UCB plasma proteome, but that further studies are needed to discern if these findings reflect causal relationships.

Melanocortin 1 Receptor Deficiency in Hematopoietic Cells Promotes the Expansion of Inflammatory Leukocytes in Atherosclerotic Mice.

Melanocortin receptor 1 (MC1-R) is expressed in leukocytes, where it mediates anti-inflammatory actions. We have previously observed that global deficiency of MC1-R signaling perturbs cholesterol homeostasis, increases arterial leukocyte accumulation and accelerates atherosclerosis in apolipoprotein E knockout (Apoe-/-) mice. Since various cell types besides leukocytes express MC1-R, we aimed at investigating the specific contribution of leukocyte MC1-R to the development of atherosclerosis. For this purpose, male Apoe-/- mice were irradiated, received bone marrow from either female Apoe-/- mice or MC1-R deficient Apoe-/- mice (Apoe-/- Mc1re/e) and were analyzed for tissue leukocyte profiles and atherosclerotic plaque phenotype. Hematopoietic MC1-R deficiency significantly elevated total leukocyte counts in the blood, bone marrow and spleen, an effect that was amplified by feeding mice a cholesterol-rich diet. The increased leukocyte counts were largely attributable to expanded lymphocyte populations, particularly CD4+ T cells. Furthermore, the number of monocytes was elevated in Apoe-/- Mc1re/e chimeric mice and it paralleled an increase in hematopoietic stem cell count in the bone marrow. Despite robust leukocytosis, atherosclerotic plaque size and composition as well as arterial leukocyte counts were unaffected by MC1-R deficiency. To address this discrepancy, we performed an in vivo homing assay and found that MC1-R deficient CD4+ T cells and monocytes were preferentially entering the spleen rather than homing in peri-aortic lymph nodes. This was mechanistically associated with compromised chemokine receptor 5 (CCR5)-dependent migration of CD4+ T cells and a defect in the recycling capacity of CCR5. Finally, our data demonstrate for the first time that CD4+ T cells also express MC1-R. In conclusion, MC1-R regulates hematopoietic stem cell proliferation and tissue leukocyte counts but its deficiency in leukocytes impairs cell migration via a CCR5-dependent mechanism.

An Early Myelosuppression in the Acute Mouse Sepsis Is Partly Outcome-Dependent.

Adult hematopoietic stem and progenitor cells (HSPCs) respond to bacterial infections by expansion to myeloid cells. Sepsis impairs this process by suppressing differentiation of stem cells subsequently contributing to an ineffective immune response. Whether the magnitude of HSPCs impairment in sepsis is severity-dependent remains unknown. This study investigated dynamics of the HSPC immune-inflammatory response in the bone marrow, splenic, and blood compartments in moribund and surviving septic mice. The 12-week-old outbred CD-1 female mice (n=65) were subjected to a cecal ligation and puncture (CLP) sepsis, treated with antibiotics and fluid resuscitation, and stratified into predicted-to-die (P-DIE) and predicted-to-survive (P-SUR) cohorts for analysis. CLP strongly reduced the common myeloid and multipotent progenitors, short- and long-term hematopoietic stem cell (HSC) counts in the bone marrow; lineage−ckit+Sca-1+ and short-term HSC suppression was greater in P-DIE versus P-SUR mice. A profound depletion of the common myeloid progenitors occurred in the blood (by 75%) and spleen (by 77%) of P-DIE. In P-SUR, most common circulating HSPCs subpopulations recovered to baseline by 72 h post-CLP. Analysis of activated caspase-1/-3/-7 revealed an increased apoptotic (by 30%) but not pyroptotic signaling in the bone marrow HSCs of P-DIE mice. The bone marrow from P-DIE mice revealed spikes of IL-6 (by 5-fold), CXCL1/KC (15-fold), CCL3/MIP-1α (1.7-fold), and CCL2/MCP-1 (2.8-fold) versus P-SUR and control (TNF, IFN-γ, IL-1β, -5, -10 remained unaltered). Summarizing, our findings demonstrate that an early sepsis-induced impairment of myelopoiesis is strongly outcome-dependent but varies among compartments. It is suggestive that the HSCPC loss is at least partly due to an increased apoptosis but not pyroptosis.

Effect of smoking on the hematopoietic stem cell count in cord blood

Effect of smoking on the hematopoietic stem cell count in cord blood

Memory of Divisional History Directs the Continuous Process of Primitive Hematopoietic Lineage Commitment.

SummaryHematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.

Three obstetric factors should be considered in umbilical cord blood donor selection

Background/Aim. The umbilical cord blood (UCB) volume and hematopoietic stem cells count are used as indicators for hematopoietic potential of UBC units. These indicators are affected by a collection method and obstetric factors. It was established that birth weight and placental weight affect the volume and hematopoietic stem cells count in UCB units. The influence of other obstetric factors is less clear. The aim of this study was to investigate the impact of obstetric factors on hematopoietic potential of UCB units. Methods. The study involved 103 consecutive UCB units collected during 2013. Relationship of UCB volume, total nucleated cells, CD34+ cells and Colony Forming Unit- Granulocyte Monocyte count with maternal and neonatal characteristics was retrospectively analyzed. Results. It was shown that birth weight, placental weight and umbilical cord length ? 31 cm significantly increased the volume of collected samples, total nucleated cells, CD34+ cells and Colony Forming Unit-Granulocyte Monocyte count. Gestational age between 38?40 weeks increased significantly all umbilical factors (volume, total nucleated cells, CD34+ cells, and Colony Forming Unit-Granulocyte Monocyte count). Gender did not have an influence on quality of UCB units except on total nucleated cells and CD34+ cells count. Other obstetric factors did not affect significantly the quality of UCB units. Conclusion. Our study confirmed that birth weight, placenta weight, length of the umbilical cord and gestational age independently influenced the UCB unit volume, and absolute count of nuclear cells and hematopoietic stem cells. Due to a positive correlation between birth weight and placental weight, only birth weight, umbilical cord length and gestational age should be standard parameters in procedure of donor selection.

Therapeutic effect of autologous bone marrow stem cell mobilization combined with anti-infective therapy on moyamoya disease.

ObjectiveThe purpose of this study is to explore the therapeutic effect of autologous bone marrow stem cell (ABMSC) mobilization combined anti-infection therapy on patients with moyamoya disease (MMD), and to provide reference for the clinical treatment of MMD and cerebrovascular disease.Methods54 adult patients with MMD diagnosed in Henan Provincial People’s Hospital from March 2017 to March 2019 were chosen as research objects. All patients were randomly divided into study group (SG) and control group (CG), with 27 patients in each group. Patients in both groups received conventional drug treatment after diagnosis of MMD, and received dura turnover of brain - temporal muscle - superficial temporal artery application surgery during indirect vascular reconstruction. On the basis of surgical treatment, patients in the SG were given ABMSC mobilization combined with low-dose dexamethasone for anti-inflammatory and anti-infection treatment. ABMSCs were mobilized by recombinant human granulocyte colony stimulating factor (rhG-csF) and recombinant human granulocyte - macrophage colony stimulating factor (rhoM-esF). The therapeutic effects of the patients were evaluated BF, one month after treatment (AF), three months AF, and six months AF. The number of hematopoietic stem cells (HpCs) and inflammatory indicators were compared between the two groups before and 4 weeks AF.ResultsFirstly, the Barthcl index of patients in the two groups showed a gradual increase trend at the 3rd and 6th months AF, and the ascensional range in the research group was higher than that in the CG (P < 0.05). Secondly, at the 3rd and 6th month AF, national institute of heath stroke scale (NIHSS) scores of patients in the CG were lower than those before treatment (BF), and there was an important change in NIHSS scores between the two groups at the same period (P < 0.05). Thirdly, after 1 month of treatment and 3 months of treatment, Chinese stroke scale (CSS) scores of patients in both groups decreased obviously compared with those BF, and the SG was lower than the CG, with statistical changes (P < 0.05). Fourthly, after 4 weeks of treatment, the hematopoietic stem cell counts in both groups were higher than those BF, and the hematopoietic stem cell counts in the SG were obviously higher than those in the CG (P < 0.05). All three inflammatory indicators were improved compared with those BF, and the SG was better than the CG (P < 0.05).ConclusionAutogenous bone marrow stem cell mobilization combined with dexamethasone anti-inflammation and anti-infection treatment after revascularization in patients with MMD can accelerate the recovery of nerve function and promote the formation of new blood vessels. At the same time, it can reduce inflammation and improve patients' quality of life, which is worthy of clinical reference.

Evaluation of the Sysmex XN automated hematopoietic progenitor cell enumeration for timing of peripheral blood stem cell harvest

BackgroundEnumeration of stem cells is essential in the management of peripheral blood stem cell (PBSC) harvest. An alternative to the gold standard flow cytometric CD34+ stem cell count is the fully automated hematopoietic stem cell (HPC) count on the Sysmex XN hematology analyzer. Materials and methodsEighty-nine patients and healthy stem cell donors who underwent PBSC harvest were included in the study. Stem cells were enumerated in pre-harvest peripheral blood and the apheresis yield by both flow cytometric CD34+ stem cell enumeration and by the Sysmex XN HPC count. ResultsThe Sysmex HPC concentration overestimated the CD34+ stem cell concentration by a ratio of 1.3 in average. The agreement between the two methods was poor at concentration <40 stem cells/μL (Bias: 45 %, 95 % limits of agreement: -71 - 160 %). CD34+ stem cell concentration and HPC concentration correlated well in pre-harvest peripheral blood (R=0.73, slope=0.96). We established a positive cut off >43.5 HPC/μL, where PBSC harvest can be initiated. And a negative cut off <16.5 HPC/μL, where harvest should be postponed or other mobilizing regimens or bone marrow harvest should be considered. 33 % of measurements were in between the negative and positive cut-off and would require a supplementary CD34+ cell count. ConclusionAlthough Sysmex HPC count correlates well with CD34+ cell count in peripheral blood, the agreement between the two methods is poor, especially at low concentrations, namely in the clinical decision range. Sysmex HPC count as a surrogate for CD34+ cell count should, therefore, be used with caution.

Clinical Evidence of Granulocyte-Monocyte Progenitor (GMP) Stem Cell Involvement in Plinabulin's Mechanism of Action (MoA) for the Prevention of Docetaxel (Doc) Chemotherapy (Chemo)-Induced Neutropenia (CIN)

Introduction: Plinabulin (Plin) is being developed as a novel non-Granulocyte colony stimulating factor (G-CSF) for CIN prevention. Previously we reported that Plin was equally effective as Pegfilgrastim for the prevention of grade 4 CIN in a Phase (Ph) 2 trial in NSCLC patients (Pts) receiving 75 mg/m2 Doc (Blayney, ASH 2017, 2018). Plin is single dose per cycle, on the same day of (and 30 min after) Chemo, does not cause bone pain and has anticancer activity. Plin is separately developed as an anti-cancer agent in a global Ph3 trial in NSCLC pts (NCT02504489). Preclinical studies showed that Plin reversed Chemo-induced inhibition of progenitor LSK differentiation in bone marrow (Ghosh, AACR 2018). We have shown that peripheral blood CD34+ hematopoietic stem cell counts increase in chemo plus plin treated patients (Blayney ASH, 2018). Since normal differentiation of GMPs leads to increases in peripheral blood myelo-monocytic (M) and granulocytic cells [neutrophils(N), eosinophils (E) and basophils (B)], we hypothesize that if GMPs are involved in Plin's CIN MoA, increases in N count would positively correlate with increases in M, E and B counts. Method: The Ph 3 portion of study BPI-2358-105 (NCT03102606) evaluated Plin (40mg) head-to-head with Pegfilgrastim (6mg) for the prevention of Doc-induced CIN in patients (pts) with NSCLC, BC or HRPC. Blood N,M,B and E counts were taken at screening, pre-and post-dose during Cycle 1 and analyzed by a central laboratory (Covance). Pts received dexamethasone (Dex) pre-medication on day (D) 0,1,2, and on D1 Docetaxel (Doc) 75 mg/m2 followed by Plin (40 mg), 30 min after completion of Doc. Here we only include data from a pre-planned interim analysis, and from the Plin arm only (n=52), and we calculated the maximum increase in N,M,E and B on day (D) 5,6,7,8,9,10 15 as % of screening counts, since N,M,E and B counts on D1-D5 are confounded by demargination and Dex effects on these counts. Results: Pearson correlation coefficients (r) with associated p-values were calculated for maximum increases in N and compared with those for M,E and B, and summarized below. Conclusions: Correlation of M, E, and B with neutrophil count increase, further confirms our proposed MoA for Plin prevention of CIN: Plin protects the neutrophil precursor from chemotherapy induced damage in bone marrow. We will further explore this MoA in other CIN studies. Table Disclosures Blayney: BeyondSpring Pharmaceuticals: Research Funding. Huang:BeyondSpring Pharmaceuticals: Employment. Mohanlal:BeyondSpring Pharmaceuticals: Employment.

Regulating HSC progenitors via cholesterol

Hematopoiesis Atherosclerosis is characterized by the buildup of cholesterol-containing lipoproteins in the vascular wall. This increased cholesterol augments hematopoietic stem and progenitor cell (HSPC) counts, and the resultant increase in leukocytes is associated with increased cardiovascular disease. Gu et al. describe a mechanism orchestrating HSPC specification from the hemogenic endothelium (HE) during embryogenesis (see the Perspective by Rajan and Berman). ApoA-I binding protein accelerated cholesterol efflux from the HE, activating the transcription factor Srebp2, which in turn transactivated Notch signaling. This mechanism also appears to be important for adult HSPC expansion in hypercholesterolemia. Science , this issue p. [1085][1]; see also p. [1041][2] [1]: /lookup/doi/10.1126/science.aav1749 [2]: /lookup/doi/10.1126/science.aaw7059

Short-Term Fasting Induces Cell Cycle Arrest in Immature Hematopoietic Cells and Increases the Number of Naïve T Cells in the Bone Marrow of Mice

Calorie restriction (CR) has been studied as a way to prolong longevity, and CR before chemotherapy can reduce hematological toxicity in cancer patients. We investigated the influence of fasting on immune cells and immature hematopoietic cells. In fasted mice, there was a significant reduction in the hematopoietic stem cell count but no significant difference for progenitor cells. Colony assays showed no difference and the rates of early and late apoptosis were almost identical when comparing fasted and control mice. DNA cell cycle analysis of immature bone marrow (BM) cells showed that CR caused a significant increase in the percentage in the G0/G1 phase and decreases in the S and G2/M phases. We detected a remarkable increase of T cells in the BM of fasted mice. CD44– naïve CD8+ T cells were more numerous in fasted BM, as were naïve CD4+ T cells, and part of those T cells showed less tendency in the G0/G1 phase. Immature hematopoietic cells remained in a relatively quiescent state and retention of colony-forming capacity during CR. The number of naïve T cells in the BM of fasted mice increased. These findings imply immature hematopoietic cells and some lymphoid cells can survive starvation, whilst maintaining their function.

AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate.

Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are unclear. Here, we report that a somite-derived prohematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which in turn transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch up-regulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing (RNA-seq), and assay for transposase-accessible chromatin using sequencing (ATAC-seq) indicate that Srebp2 transregulates Notch pathway genes required for hematopoiesis. Our studies outline an AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease.

Effects of High- and Low-LET Radiation on Human Hematopoietic System Reconstituted in Immunodeficient Mice

Over the last 50 years, a number of important physiological changes in humans who have traveled on spaceflights have been catalogued. Of major concern are the short- and long-term radiation-induced injuries to the hematopoietic system that may be induced by high-energy galactic cosmic rays encountered on interplanetary space missions. To collect data on the effects of space radiation on the human hematopoietic system in vivo, we used a humanized mouse model. In this study, we irradiated humanized mice with 0.4 Gy of 350 MeV/n 28Si ions, a dose that has been shown to induce tumors in tumor-prone mice and a reference dose that has a relative biological effectiveness of 1 (1 Gy of 250-kVp X rays). Cell counts, cell subset frequency and cytogenetic data were collected from bone marrow spleen and blood of irradiated and control mice at short-term (7, 30 and 60 days) and long-term ( 6 - 7 months) time points postirradiation. The data show a significant short-term effect on the human hematopoietic stem cell counts imparted by both high- and low-LET radiation exposure. The radiation effects on bone marrow, spleen and blood human cell counts and human cell subset frequency were complex but did not alter the functions of the hematopoietic system. The long-term data acquired from high-LET irradiated mice showed complete recovery of the human hematopoietic system in all hematopoietic compartments. The combined results demonstrate that, in spite of early perturbation, the longer term effects of high-LET radiation are not detrimental to human hematopoiesis in our system of study.

Targeting NAD+ Modulating Enzymes to Improve HSC Function in Aging and Bone Marrow Failure

Human hematopoietic system produces various types of differentiated and short-lived cells with specialized functions, which require continuous replenishment through the function of hematopoietic stem cells (HSC). HSC failure is a common distal endpoint of various pathogenic mechanisms in almost all bone marrow failure (BMF) syndromes and associated diseases. Hematopoietic growth factor cocktails (HGF) used in expanding bone marrow cells e.g., to increase cellularity of the HSC grafts, lead to differentiation and decreased HSC count. Theoretically, when used in vivo, they may act on progenitors rather than HSC and lead to stimulation of clonal outgrowth. Our current ability to stimulate HSC self-renewal to provide reconstitution of long-term hematopoiesis is limited.Nicotinamide adenine dinucleotide (NAD+) serves as an essential cofactor and substrate for a number of critical cellular processes. NAD+ depletion may occur in response to DNA damage due to free radical/ionizing radiation attack, resulting in significant activation of NAD+ consuming PARPs. Because of their long lifespan, maintenance of the genomic integrity of HSCs by efficient and accurate DNA repair to reduce the risk of BMF and cellular transformation is essential. NAD+ is also required for the maintenance of sirtuins activity, important class III HDAC essential for the prevention of senescence. Aging or chronic immune activation and inflammatory cytokine production result in upmodulation of NAD+ degrading enzyme CD38 that rapidly depletes cellular and extracellular levels of NAD+. Various lines of evidence suggest that regulation of CD38 NADase activity is essential for maintenance of physiologic NAD+ levels. Enhancing NAD+ level can profoundly reduce oxidative cell damage in catabolic tissue, including blood. Consequently, promotion of intracellular NAD+ by preventing NAD+ catabolism represents a promising therapeutic strategy for degenerative diseases in general, and BMF and associated diseases in particular. Therefore; CD38, a major NAD+ degrading enzyme, can be an excellent therapeutic target to increase the cellular levels of NAD+ and consequently improve the function of HSC. Here we report the development of inhibitors of CD38 NADase activity that extends the self-renewal and proliferative life span of HSC.We used structure-guided virtual screening followed by docking simulation to develop CD38 inhibitors. The compounds were synthesized using rational chemical synthesis and characterized by high-resolution mass spectroscopy and C13 & H1NMR. HPLC based assays were performed to assess the ability of compounds to inhibit NAD+ degradation by recombinant CD38. Using an iterative approach of synthesis characterization and activity, we selected the most potent compound, designated as ccf1172, for further studies. Docking simulations, surface plasmon resonance, and HPLC based assays demonstrate that ccf1172 binds (KD=12 nM) and inhibits CD38 (IC50=10 nM) (Fig.1B, C&D). To further characterize the ability of ccf1172, colony forming assays (CFU-A) and long-term culture-initiating cell assays (LTCIC-A) were performed with cord blood, human and murine bone marrows. No GF-like activity was observed, but in combination with GF mix ccf1172 increased the number of erythroid and myeloid colonies (n=9) in dose-dependent manner with a maximal effect seen at 100 nM in a serial replating assay. Significant extension of proliferative life span of hematopoietic progenitors (n=5) were observed (Fig 1E). When we studied the ability of CD38 inhibitor to expand LTCICs in stromal cultures (n=3) as best in vitro surrogates of HSC, ccf1172 increased LTCIC numbers 2.6-fold at 10 nM. The effect did not require the presence of accessory cells as ccf1172 treatment resulted in ~2-fold increase in CD34+Lin-/CD45+ cells in stem cell culture media supplemented with growth factors over a period of 25 days (Fig 1F). The CD38 inhibitor demonstrated cytotoxic effects on nine different leukemic cell lines with IC 50 ranging from 1 to 5 µM while no effect was observed on normal bone marrow.Here, we demonstrate that CD38 inhibition may be a potential therapeutic principle for ex vivo and in vivo expansion of HSC. Decreasing levels of NAD+ have been linked to aging and stem cell dysfunction, as a key aspect of various BMF syndromes. The strategy of CD38 inhibition to preserve NAD+ is innovative and relevant therapeutic strategy. [Display omitted] DisclosuresSaunthararajah:Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy.

CD34+ Hematopoietic Stem Cell Count Is Predictive of Vascular Event Occurrence in Children with Sickle Cell Disease.

Sickle cell disease (SCD) complications mostly result from vascular dysfunction, concerning systemic microvasculature and cerebral large vessels. The aim of this cohort study was to identify potential circulating biomarkers predictive for further vascular event occurrence in pediatric SCD. We consecutively enrolled 108 children with SCD at steady state, aged 3-18years old (median 9.8years). Hematology, coagulation, hemolysis, endothelial, platelet and vascular activation parameters were recorded at inclusion. Neurovascular and systemic vascular events were prospectively recorded during a mean follow-up period of 27months. Patients at steady state displayed significantly higher hemolysis and platelet activation markers, higher leukocyte, CD34+ hematopoietic stem cell and microvesicle counts, and a pro-coagulant profile compared to controls matched for age and ethnicity. Circulating endothelial cell or nucleosome level did not differ. During the follow-up period, 36 patients had at least one neurovascular (n = 12) or systemic vascular event (n = 25). In a multivariate model, high CD34+ cell count was the best predictor for the occurrence of a vascular event (OR 1.2 for 1000 cell/mL increase, 95% CI [1.049-1.4], p = 0.013, sensitivity 53%, specificity 84% for a threshold of 8675 cells/mL). CD34+ cell count at steady state is a promising biomarker of further vascular event in children with SCD.

Radioprotective effects of dammarane sapogenins against 60 Co-induced myelosuppression in mice.

Radiotherapy frequently induces failure of hematopoietic system and leads to myelosuppression. The objective of this study was to investigate the protective effect of dammarane sapogenins (DS), the hydrolysed product of the constituent ginsenosides of Panax ginseng, which are produced by gut metabolism, on radiation-induced hematopoietic injury. Mice were exposed to 3.5Gy 60 Co γ-rays of total body radiation at a dose rate of 1.60Gy per minute and treated with DS or granulocyte colony-stimulating factor immediately after radiation. The general condition of the mice, the peripheral blood cell counts, multiple colony forming unit (CFU) assays of hematopoietic progenitor cells, hematopoietic stem cell counts, bone marrow histology, and spleen colony forming unit counts were then investigated. Our results indicated that administration with DS could ameliorate 60 Co-irradiation induced damage and significantly increase the number of peripheral blood cells (white blood cells and platelets), 5 types of hematopoietic progenitor cells CFU (CFU-GM, CFU-E, BFU-E, CFU-Meg, and CFU-GEMM), hematopoietic stem cell (Lin- c-kit+ Scal-1+ ) numbers, and CFUs in the spleen, as well as improved bone marrow histopathology. All together, these results confirmed the enhancement of DS on hematopoiesis.