Peripheral Blood-derived CD34 Research Articles

Increased peritoneal TGF-β1 is associated with ascites-induced NK-cell dysfunction and reduced survival in high-grade epithelial ovarian cancer.

Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cell-mediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-β1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-β1 exposure. Moreover, inhibition of TGF-β1 signaling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-β1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-β1 signaling could increase NK cell immune responses in high-grade EOC patients.

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

A Clinical Trial of Comprehensive Regenerative Treatment for Acute and Chronic Kidney Injury by Autologous Granulocyte-Colony Stimulating Factor (G-CSF)-Mobilized Peripheral Blood-Derived CD34+ Cell Administration

A Clinical Trial of Comprehensive Regenerative Treatment for Acute and Chronic Kidney Injury by Autologous Granulocyte-Colony Stimulating Factor (G-CSF)-Mobilized Peripheral Blood-Derived CD34+ Cell Administration

Apoptosis Evaluation in Circulating CD34+-Enriched Hematopoietic Stem and Progenitor Cells in Patients with Abnormally Increased Production of Endogenous Glucocorticoids in Course of Cushing's Syndrome.

Abnormalities in hematological parameters of peripheral blood have been noted in patients with endogenous Cushing's Syndrome (CS) in the corticotropin (ACTH)-dependent and ACTH-independent forms. Nevertheless, the exact mechanism of glucocorticoids (GCs) action on human hematopoiesis is still not entirely clear. The aim of the study was to determine whether endogenous excessive production of GCs could affect apoptosis of CD34+ cells enriched in hematopoietic stem and progenitor cells (HSPCs) collected from the peripheral blood of newly diagnosed CS patients. Flow cytometry, Annexin-V enzyme-linked immunosorbent assay, TUNEL assay, real-time quantitative PCR, and microarray RNA/miRNA techniques were used to characterize CS patients' HSPCs. We found that the glucocorticoid receptor (GR) protein expression levels in CS were higher than in healthy controls. A complex analysis of apoptotic status of CS patients' HSPC cells showed that GCs significantly augmented apoptosis in peripheral blood-derived CD34+ cells and results obtained using different methods to detect early and late apoptosis in analyzed cell population were consistent. CS was also associated with significant upregulation in several members of the BCL-2 superfamily and other genes associated with apoptosis control. Furthermore, global gene expression analysis revealed significantly higher expression of genes associated with programmed cell death control in HSPCs from CS patients. These findings suggest that human endogenous GCs have a direct pro-apoptotic activity in hematopoietic CD34+ cells derived from CS subjects before treatment.

Tenofovir disoproxil fumarate-mediated γ-globin induction is correlated with the suppression of trans-acting factors in CD34+ progenitor cells: A role in the reactivation of fetal hemoglobin

Sickle-cell disease (SCD) and β-thalassemia are public health issues that affect people all over the world. Fetal hemoglobin (HbF) induction is a molecular intervention, including hydroxyurea, which has made an effort to improve current treatment. Tenofovir disoproxil fumarate (TDF) is formerly reported with improving levels of hemoglobin, mean corpuscular hemoglobin (MCH), and mean corpuscular volume (MCV). Hence, in this preclinical investigation, human peripheral whole blood-derived CD34+ progenitor cells were cultured to prove the efficacy of TDF on erythroid proliferation, differentiation, γ-globin gene expression regulation, and ultimately HbF production. We observed that TDF increased the proliferation of immature erythroid cells, delayed the terminal erythroid maturation without cytotoxicity as correlated with other HbF inducers. Here, the presented data show that TDF can induce HbF expression by up-regulating the γ-globin gene transcription up to 7.1 ± 0.46-fold and subsequently increased the F-cells (10.79 ± 1.9-fold) population in terminally differentiated erythroid cells. Furthermore, our findings demonstrated that TDF-mediated γ-globin gene induction and HbF production was associated with down-fold regulation of BCL11A and SOX6, and their corresponding trans-acting regulators, FOP, KLF1, and GATA1. Collectively, our findings suggest TDF as an effective inducer of HbF in CD34+ cells and pave the way to put forward the assessment of TDF as a new potential therapy in treating β-hemoglobinopathies.

A Pharmacologic Approach Using Pomalidomide and Dexamethasone to Study Stage-Specific Regulation of Erythroid Progenitors

The erythroid progenitors, burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E), control red cell production through the integration of a variety of self-renewal and differentiation signals. The identification of these signals and their effects on erythroid progenitor populations are of therapeutic interest for human disorders characterized by decreased red cell production such as Diamond Blackfan Anemia (DBA). We have previously documented that pomalidomide acts at the BFU-E stage and dexamethasone at the CFU-E stage in in vitro models of human erythropoiesis. Therefore, these two agents represent potential pharmacologic interventions for the treatment of human diseases with disordered erythropoiesis. Here, we studied the mechanisms by which pomalidomide and dexamethasone enhance erythroid progenitor numbers in cord blood and adult bone marrow-derived CD34+ hematopoietic stem and progenitor cells (HSPC).Pomalidomide increased adult and fetal BFU-E numbers in both serum-free and serum-containing in vitro erythroid culture systems. CFU-E numbers and terminal erythroblasts were also transiently attenuated suggesting that pomalidomide mediates increased BFU-E in part through delayed progression to erythropoietin-sensitive progenitors and precursor stages. Transforming growth factor-β (TGF-β) has previously been shown to regulate the transition from BFU-E to CFU-E, and therefore, we investigated whether pomalidomide modulates TGF-β signaling. We noted accelerated differentiation as assessed by glycophorin A (GPA) expression of CD34+ HSPCs treated with TGF-β1, and the addition of pomalidomide completely abrogated TGF-β1 mediated differentiation. This effect was not due to inhibition of TGF-β1 receptors or kinases as treatment of NIH 3T3 cells with TGF-β1 ± pomalidomide failed to prevent SMAD2 phosphorylation. Only CD34+ HSPCs differentiated in the presence of pomalidomide exhibited reduced sensitivity to TGF-β1. These results suggest that pomalidomide alters erythroid and/or earlier hematopoietic progenitor differentiation to enrich a population of TGF-β1 insensitive cells and/or affects the activity of downstream TGF-β1 effectors.Dexamethasone, in contrast to pomalidomide, was found to specifically increase the number of CFU-E progenitors. Elevated CFU-E self-renewal was associated with an accumulation of cells in G0/G1 of the cell cycle, which suggests that alterations to cell cycle progression may contribute to the regulation of self-renewal genes in CFU-E. In contrast to adult-derived CFU-E, dexamethasone failed to affect CFU-E numbers or cell cycle status in cord blood derived CFU-Es. We investigated the expression of glucocorticoid receptor isoforms (GRα and GRβ) in erythroid progenitors, which revealed that BFU-E and CFU-E differentially express GR isoforms. BFU-E expressed comparable levels of GRα and the hypothesized dominant-negative GRβ, where as only GRα transcript was measured in CFU-E. These expression patterns were similar between adult and cord blood-derived progenitors. Despite similar GR levels, adult-derived erythroid progenitors, but not cord blood derived progenitors, were shown to translocate GRα to the nucleus after a 24hr pulse of dexamethasone suggesting that post-translational mechanisms may govern dexamethasone response in erythroid progenitors.These findings strongly indicate that pharmacologic modulation of the TGF-β and glucocorticoid-signaling pathways can effectively stimulate erythroid progenitor self-renewal. We are currently elucidating specific targets for pomalidomide and dexamethasone in BFU-E and CFU-E, respectively, through proteomics and transcriptome approaches, and investigating the efficacy of mono- and combination therapies using peripheral blood-derived CD34+ HSPCs from DBA patients to study their therapeutic efficacy. DisclosuresNo relevant conflicts of interest to declare.

Simultaneous Activation of Signals Through gp130, c-kit, and Interleukin-3 Receptor Promotes a Trilineage Blood Cell Production in the Absence of Terminally Acting Lineage-Specific Factors

AbstractWe assessed the biologic role of signaling through gp130, a signal-transducing receptor (R) component, in human hematopoiesis in vitro. Although peripheral blood-derived CD34+ cells ubiquitously expressed gp130 and interleukin-3 receptor α (IL-3Rα), IL-6Rα was only detected on 80% of these CD34+ cells. We sorted CD34+IL-6R+ or CD34+IL-6R− cells and studied the effect on hematopoietic colony formation of signaling through gp130 activated by IL-6 or a combination of IL-6 and recombinant soluble human IL-6R (sIL-6R) in the presence or absence of stem cell factor (SCF ) and/or IL-3. Signals activated by SCF, IL-6, or IL-6/sIL-6R complex alone did not induce significant colony formation. However, a combination of IL-3, SCF, and IL-6/sIL-6R complex dramatically induced many neutrophil (colony-forming unit-granulocyte [CFU-G]), erythroid burst (burst-forming unit-erythrocyte [BFU-E]), erythrocyte-containing mixed (CFU-Mix), and megakaryocyte (CFU-Meg) colony formations when CD34+IL-6R− cells were used as the target. CFU-G colony formation induced by the three signals was more evident when CD34+IL-6R+ cells were used as the target. This distinct synergistic effect of the three different signals was confirmed by single-cell clone-sorting experiments. Moreover, colony formation (including CFU-G, BFU-E, CFU-Mix, and CFU-Meg) was observed even in the presence of neutralizing antibodies for granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin (c-Mpl), whereas neutralizing antibodies for gp130, IL-6R, IL-3, and SCF partially or completely blocked the synergistic effect. The maturation of neutrophilic, erythroid, and megakaryocytic cells supported by the three signals in serum-free cultures was confirmed by immunostaining using anti-CD66b, antiglycophorin A, antihemoglobin α, and anti-CD41 monoclonal antibodies, respectively. In contrast, any two of the three signals were insufficient for effective blood cell production in the absence of maturation factors. These results suggest that simultaneous activation of the three signals through gp130, c-kit, and IL-3R can induce in vitro proliferation and differentiation of trilineage hematopoietic progenitors in the absence of terminally acting lineage-specific factors.

Targeting of Calbindin 1 (CALB1) Rescues Erythropoiesis in a Human Model of Diamond Blackfan Anemia: Implications for Novel Therapies

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by red cell aplasia, congenital anomalies and a predisposition to cancer. The erythroid defects of DBA include macrocytosis, reticulocytopenia, and a selective decrease or absence of erythroid precursors in the bone marrow. It was the first disease linked to ribosomal dysfunction and >70% of patients have since been shown to have a haploinsufficiency of a ribosomal protein (RP) gene. Among these RP mutations, RPS19 mutations were the first to be identified and are the most common, found in up to 25% of DBA patients. Current therapies for DBA include corticosteroids, chronic transfusions, and/or hematopoietic stem cell transplantation. However, each of these therapies have potential for significant side effects and as such there is an urgent need to develop new therapies for the management of these patients. In addition there is a need for improved understanding of the mechanistic basis for the wide range of phenotypic expression and responses to therapy between patients with the same mutation, which implies a key role for modifier genes in this disease. To identify potential modifiers in the phenotype of patients and for developing potential novel therapeutic targets, we performed a genome wide CRISPR screen using A549 cells with RPS19 haploinsufficiency as a cellular model for DBA, which enabled large-scale screening. As the p21 pathway is known to be central to the pathophysiology of DBA, we identified hits that resulted in higher and lower levels of p21 expression in the DBA model vs. control, respectively. Among these hits, we consistently identified Calbindin 1 (CALB1), a member of the calcium-binding superfamily that includes calmodulin and buffers entry of calcium into cells upon stimulation of glutamate receptors. Given the recent findings that calmodulin inhibition can rescue anemia in DBA models by blocking the p90 ribosomal S6 kinase (RSK)-p70 (p70S6K) pathway and the role for glucose and glutamine metabolism in the regulation of human HSC lineage specification, we decided to pursue CALB1 as a potential target of interest. We used peripheral blood derived-CD34+ cells placed in erythroid stimulating media following knockdown of RPS19, and investigated the effects of CALB1 on cell cycle progression and erythroid differentiation by flow cytometry. We observed that knockdown of CALB1 significantly improved cell viability, increased G0/G1 population and promoted erythroid maturation with increased expression of CD36 and glycophorin A in the context of RPS19 deficiency. Together, these data suggest that the knockdown of CALB1 rescues human erythropoiesis in a model of DBA. We are investigating the effects of CALB1 knockdown on signaling pathways and genes involved in DBA due to RPS19 mutations notably GATA-1/HSP70 and TNF-alpha. We anticipate that the identification of a role for CALB1 in the pathophysiology of DBA will provide a new therapeutic target for DBA either alone or in combination with steroids or calmodulin inhibition. Disclosures Glader: Agios Pharmaceuticals, Inc.: Consultancy.

UM171 promotes expansion of autologous peripheral blood hematopoietic stem cells from poorly mobilizing lymphoma patients

BackgroundAutologous hematopoietic stem cell transplantation is an effective therapeutic strategy for lymphoma patients. However, some patients have to give up receiving transplantation because of failing to obtain sufficient CD34+ cells yields. Therefore, we ex vivo expanded HSCs of lymphoma patients using UM171 to solve the problem of HSCs deficiency. MethodsMobilized peripheral blood-derived CD34+ cells from lymphoma patients were cultured for 10 days with or without UM171. The fold of cell expansion and the immunophenotype of expanded cells were assessed by flow cytometry. RNA-seq experiment was performed to identify the mechanism by which UM171 promoted HSCs expansion. ResultsUM171 treatment increased the proportion of CD34+ (68.97 ± 6.91%), CD34+ CD38− cells (44.10 ± 9.20%) and CD34+CD38−CD45RA−CD90+ LT-HSCs (3.05 ± 2.08%) compared to vehicle treatment (36.08 ± 11.14%, 18.30 ± 9.49% and 0.56 ± 0.45%, respectively). UM171 treatment led to an 85.08-fold increase in LT-HSC numbers relative to initial cells. Importantly, UM171 promoted expansion of LT-HSCs achieved 138.57-fold in patients with poor mobilization. RNA-seq data showed that UM171 upregulated expression of HSC-, mast cell-specific genes and non-canonical Wnt signaling related genes, and inhibited genes expression of erythroid, megakaryocyte and inflammatory mediated chemokine. ConclusionsOur study shows that UM171 can efficiently promote ex vivo expansion of HSCs from lymphoma patients, especially for poorly mobilizing patients. In terms of mechanism, UM171 upregulate HSC-specific genes expression and suppress erythroid and megakaryocytic differentiation, as well as activate non-classical Wnt signaling.

Glucocorticoids Induce the Maintenance and Expansion of an Immature CFU-E Erythroid Progenitor Population in Humans

Glucocorticoids are widely used to increase red cell production in a number of hematological disorders including Diamond Blackfan anemia (DBA). Despite their clinical effectiveness we do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. To improve the clinical management of these patients, we studied the mechanisms of dexamethasone (Dex) on human erythroid progenitors. We employed an erythroid culture system to differentiate primary human CD34+ cells isolated from patients with DBA and healthy controls as well as cord blood samples. We found that Dex increases the proliferation of CD34+ cells derived from peripheral blood but surprisingly not of CD34+ cells from cord blood. Furthermore, by mass spectrometry proteomic analysis, we found that Dex specifically altered the induction of several targets unique to peripheral blood including the transcription factor and cell cycle regulator NR4A1. When examining the cell populations expanded with Dex treatment by immunophenotyping, we detected a specific expansion of CFU-Es. Dex increased the average size of CFU-E colonies in methylcellulose colony forming assays in the presence of Epo alone, suggesting that CFU-Es are the most Dex responsive progenitor in humans. In support of this hypothesis, reticulocyte counts from patients with DBA treated with prednisone were found to increase approximately one week after treatment, consistent with the time course of differentiation of CFU-E to reticulocytes in bone marrow. In order to further resolve the progenitor target of steroid responsive progenitor we developed a new gating scheme that allows for increased resolution of BFU-E and CFU-E subpopulations by monitoring surface expression of CD71 and CD105. Using this system, we found that Dex treatment led to the maintenance of a novel immature transitional progenitor population in peripheral blood-derived cells that we term immature CFU-E. In methylcellulose colony forming assays, these peripheral blood-derived immature CFU-E treated with Dex produced larger colonies than the untreated controls and mature CFU-E. The expansion and proliferation of Dex treated immature CFU-Es derived from peripheral blood was associated with a decreased percentage of cells in the S phase of the cell cycle. No change was noted for either Dex treated BFU-E from peripheral blood or for BFU-E or CFU-E from cord blood. Importantly, Dex responsive peripheral blood-derived CFU-Es divided at a faster rate than untreated CFU-Es, revealing a dissociation in the link between differentiation and proliferation. Mechanistically, we found that Dex treated peripheral blood CFU-E exhibited increased expression of the negative cell cycle regulator p57Kip2. Moreover, this protein was also found to be dysregulated in CD34+ cells from transfusion-dependent patients with DBA, wherein p57Kip2 expression was not significantly altered following Dex treatment, indicating a role for p57Kip2 in the erythroid progenitor defects seen in these patients. To further evaluate the role of p57Kip2 in the Dex dependent alteration in CFU-E proliferation, peripheral blood-derived CD34+ cells were transduced with p57Kip2-specific shRNA lentiviral constructs. shRNA knockdown of p57Kip2 abrogated the effects of Dex on growth and induced a premature EPO-mediated differentiation. In summary, our findings provide new insights into functional heterogeneity of human erythroid progenitors and reveal mechanistic insights into the action of Dex in normal and disordered erythropoiesis. Disclosures Vlachos: Novartis: Other: Steering committee member.

Echinochrome A Promotes Ex Vivo Expansion of Peripheral Blood-Derived CD34+ Cells, Potentially through Downregulation of ROS Production and Activation of the Src-Lyn-p110δ Pathway.

Intracellular reactive oxygen species (ROS) play an important role in the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). HSPCs are difficult to be expanded ex vivo while maintaining their stemness when they are exposed to oxidative damage after being released from the bone marrow. There have been efforts to overcome this limitation by using various cytokine cocktails and antioxidants. In this study, we investigated the effects of echinochrome A (Ech A)-a well-established and non-toxic antioxidant-on the ex vivo expansion of HSPCs by analyzing a CD34+ cell population and their biological functions. We observed that Ech A-induced suppression of ROS generation and p38-MAPK/JNK phosphorylation causes increased expansion of CD34+ cells. Moreover, p38-MAPK/JNK inhibitors SB203580 and SP600125 promoted ex vivo expansion of CD34+ cells. We also demonstrated that the activation of Lyn kinase and p110δ is a novel mechanism for Ech A to enhance ex vivo expansion of CD34+ cells. Ech A upregulated phospho-Src, phospho-Lyn, and p110δ expression. Furthermore, the Ech A-induced ex vivo expansion of CD34+ cells was inhibited by pretreatment with the Src family inhibitor PP1 and p110δ inhibitor CAL-101; PP1 blocked p110δ upregulation and PI3K/Akt activation, whereas CAL-101 and PI3K/Akt pathway inhibitor LY294002 did not block Src/Lyn activation. These results suggest that Ech A initially induces Src/Lyn activation, upregulates p110δ expression, and finally activates the PI3K/Akt pathway. CD34+ cells expanded in the presence of Ech A produced equal or more hematopoietic colony-forming cells than unexpanded CD34+ cells. In conclusion, Ech A promotes the ex vivo expansion of CD34+ cells through Src/Lyn-mediated p110δ expression, suppression of ROS generation, and p38-MAPK/JNK activation. Hence, Ech A is a potential candidate modality for the ex vivo, and possibly in vivo, expansion of CD34+ cells.

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

BackgroundDisease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool for elucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patient peripheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimum invasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitor cells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally require HSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogramming efficiencies, making the overall procedure costly, laborious, and time-consuming.MethodsWe have established a highly efficient method for generating iPSCs from non-mobilized PB-derived CD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and were kept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads, with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vector SeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generation series, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time were recorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cell receptor gene rearrangement, pluripotency markers, and differentiation capability were examined.ResultsWe succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay.ConclusionThis robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases.

Reassessing the effects of continuous positive airway pressure (CPAP) on arterial stiffness and peripheral blood derived CD34+ progenitor cells in subjects with sleep apnea

BackgroundObstructive sleep apnea (OSA) is an independent risk factor for cardiovascular diseases (CVD) and vascular health. Peripheral blood-derived CD34+ progenitor cells have been used as biomarker for CVD risk and may play a similar role in OSA and CVD risk assessment. Although there are some controversial results in the literature, OSA patients may have a reduction in the number and function of CD34+ cells. The damages promoted by OSA in CD34+ cells may lead to an increase in endothelial oxidative stress and endothelial inflammation which may lead to a reduced endothelial repair capacity. In this study, we explored the effect of continuous positive airway pressure (CPAP) on peripheral blood-derived CD34+ cells and arterial stiffness (another predictor of endothelial health and CVD risk) in OSA patients.Methods and resultsNine overweight and obese subjects without prediabetes or diabetes were recruited. Eight out of nine subjects had moderate to severe degree of OSA. CD34+ cells were isolated from peripheral blood. Number and function of these cells were monitored before and after 3 months of treatment with CPAP. No significant changes were observed in the number of CD34+ cells, CFU-Hill’s colony formation unit (CFU) count or migratory response to the chemotactic factor SDF-1a after CPAP use. However, CXCR4 mRNA expression significantly increased by 2.2-fold indicating that CPAP may have a positive effect on SDF1a receptor (CXCR4), thereby improving migration of CD34+ cells mediated by SDF1a after the 3 month period. Interestingly, in clinical arena our results showed a reduction of pulse wave velocity (an established parameter of arterial stiffness) following CPAP therapy.ConclusionsOur findings suggest that 3-month CPAP intervention does not show statistical significant increase in CD34+ cell number and function, in mostly moderate to severe OSA subjects; however, it did demonstrate a positive trend. CPAP therapy, did help improve arterial stiffness parameter.

Design and Validation of an Automated Process for the Expansion of Peripheral Blood-Derived CD34+ Cells for Clinical Use After Myocardial Infarction.

We previously demonstrated that intracardiac delivery of autologous peripheral blood‐derived CD34+ stem cells (SCs), mobilized by granulocyte‐colony stimulating factor (G‐CSF) and collected by leukapheresis after myocardial infarction, structurally and functionally repaired the damaged myocardial area. When used for cardiac indication, CD34+ cells are now considered as Advanced Therapy Medicinal Products (ATMPs). We have industrialized their production by developing an automated device for ex vivo CD34+‐SC expansion, starting from a whole blood (WB) sample. Blood samples were collected from healthy donors after G‐CSF mobilization. Manufacturing procedures included: (a) isolation of total nuclear cells, (b) CD34+ immunoselection, (c) expansion and cell culture recovery in the device, and (d) expanded CD34+ cell immunoselection and formulation. The assessment of CD34+ cell counts, viability, and immunophenotype and sterility tests were performed as quality tests. We established graft acceptance criteria and performed validation processes in three cell therapy centers. 59.4 × 106 ± 36.8 × 106 viable CD34+ cells were reproducibly generated as the final product from 220 ml WB containing 17.1 × 106 ± 8.1 × 106 viable CD34+ cells. CD34+ identity, genetic stability, and telomere length were consistent with those of basal CD34+ cells. Gram staining and mycoplasma and endotoxin analyses were negative in all cases. We confirmed the therapeutic efficacy of both CD34+‐cell categories in experimental acute myocardial infarct (AMI) in immunodeficient rats during preclinical studies. This reproducible, automated, and standardized expansion process produces high numbers of CD34+ cells corresponding to the approved ATMP and paves the way for a phase I/IIb study in AMI, which is currently recruiting patients. stem cells translational medicine 2019;8:822&832

Rapid and Efficient Production of Human Functional Mast Cells through a Three-Dimensional Culture of Adipose Tissue-Derived Stromal Vascular Cells.

Mast cells (MC) are innate immune cells involved in many physiological and pathological processes. However, studies of MC function and biology are hampered by the difficulties to obtain human primary MC. To solve this problem, we established a new method to produce easily and rapidly high numbers of MC for in vitro studies using human adipose tissue, which is an abundant and easy access tissue. Stromal vascular fraction of adipose tissue, obtained from human abdominal dermolipectomy, was cultured as spheroids in serum free medium supplemented in stem cell factor. Using this method, we generated, within 3 wk, a highly pure population of connective tissue-type MC expressing MC typical peptidases (tryptase, chymase, and carboxypeptidase-A3) with a yield increasing over time. Stem cell factor was required for this culture, but unlike MC derived from CD34+ cells, this culture did not depend on IL-3 and -6. MC obtained with this method degranulated following FcεRI cross-linking or stimulation by C5a, compound 48/80, and substance P. Interestingly, activation by anti-IgE of both white adipose tissue-MC and MC obtained from peripheral blood-derived CD34+ pluripotent progenitor cells induced the production of PGs as well as proinflammatory cytokines (TNF-α, Il-6, and GM-CSF). In conclusion, we developed a new time saving and reproducible method to produce highly pure and functional human MC in 3 wk from human adipose tissue.

Human mast cell neutral proteases generate modified LDL particles with increased proteoglycan binding

Background and aimsSubendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. MethodsMature human MCs were differentiated from human peripheral blood-derived CD34+ progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. ResultsActivated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. ConclusionsThe present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention.

Calmodulin Inhibition Rescues DBA Models with Ribosomal Protein Deficiency through Reduction of RSK Signaling

Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations are thought to cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29, a ribosomal gene found mutated in DBA patients. Rps29-/- embryos have hematopoietic and endothelial defects, including decreased cmyb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we tested them in human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19, the CaM inhibitor trifluoperazine (TFP) relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels and reduced p53 activity in the bone marrow. Of note, the effect of TFP was specific to RP deficiency and had no effect on erythroid differentiation or p53 activity in WT cells or mice. This prompted us to hypothesize that TFP is selectively blocking signaling in the RP deficient state, possibly through inhibition of CaM-dependent kinases. In vitro kinase profiling of over 100 kinases revealed that TFP and other positive hits from our screen inhibited the activity of p70 ribosomal S6 kinase (p70S6K) and multiple members of p90 ribosomal S6 kinase (RSK) family. RSKs are highly conserved serine/threonine kinases that regulate cell growth, migration, and survival. They can activate mTOR and directly phosphorylate RPS6. Strikingly, RSK protein levels are elevated in lysates from rps29-/- embryos and treatment with RSK or p70S6K inhibitors increased Hb in rps29-/- embryos in vivo, mimicking CaM inhibitors. RSK phosphorylation is also increased in human RPS19-deficient peripheral blood-derived CD34+ cells compared to WT cells. Treatment with TFP reduced RSK phosphorylation and decreased signaling downstream of RSK, including p70S6K, but only in the presence of RP deficiency. Similarly, TFP reduced p53 and phosphorylation of p53 at S392, but only in the presence of RP deficiency. An in vitro kinase assay determined that p70S6K directly phosphorylated p53 at S392 but not other commonly modified residues, S15 and S20. Mass spectrometry analysis of posttranslational modifications of p53 isolated from TFP treated cells revealed a significant reduction of peptides that contain phosphorylated S392. We hypothesized that TFP inhibited phosphorylation of S392. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a negative control transactivation domain mutant, p53-S15D, resulted in a 4-fold reduction in p21 mRNA levels, while cells containing p53-S392D had no reduction in p21 mRNA in response to TFP. In conclusion, we have shown that RP deficiency increases RSK phosphorylation and CaM inhibitors decrease signaling downstream of RSK, which leads to a reduction of p53 activity and rescues the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP for 2017. DisclosuresZon:Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

TRPV4 Mediates Mast Cell Activation in Cathelicidin-Induced Rosacea Inflammation

TRPV4 Mediates Mast Cell Activation in Cathelicidin-Induced Rosacea Inflammation

Growth of human mast cells from bone marrow and peripheral blood-derived CD34(+) pluripotent hematopoietic cells.

Human mast cells (HuMCs) are derived from CD34(+) pluripotent hematopoietic cells which are KIT (CD117)(+) and FcεRI(-), and lack lineage-specific surface markers. Bone marrow and peripheral blood are the two readily available sources for obtaining CD34(+) cells from which HuMCs can be cultured. CD34(+) cells are isolated and enriched by magnetic separation columns and stored under specific conditions until ready for use. Alternatively, enriched CD34(+) cells may be immediately cultured in serum-free culture media containing recombinant human (rh) stem cell factor (SCF), rhIL-6, and rhIL-3 (added only during the first week). Weekly hemidepletions and removal of adherent cells and/or debris enables the investigator to obtain HuMC cultures, identified by Wright-Giemsa and acidic toluidine blue stains, by 8-10 weeks.

Abstract 19049: MiR-126 Containing Exosomes From Human CD34+ Stem Cells Activate Endothelial Cells to Initiate Angiogenesis and Ischemic Tissue Repair

Introduction: Locally transplanted human CD34+ stem cells have been shown to improve exercise tolerance in patients with myocardial ischemia and promote angiogenesis in animal models. Recently we have demonstrated that CD34+ cells secrete membrane bound nano-vesicles called exosomes as a major component of their pro-angiogenic paracrine secretion. We hypothesize that cell-free exosomes from CD34+ cells (CD34 Exo) mimic the beneficial effects of the cells and promote angiogenesis and ischemic tissue repair via transfer of pro-angiogenic microRNAs, possibly to endothelial cells. Methods and Results: Therapeutic potential of CD34 Exo isolated from equal number of adult human peripheral blood-derived CD34+ cells was evaluated in an immunodeficient murine model of hind limb ischemia. Similar to cells, treatment with CD34 Exo resulted in significant improvement in ischemia compared to treatment with PBS (perfusion ratio: 1.01±0.04 v 0.57±0.1; capillary density: 1.8±0.3 v 0.9±0.1/HPF; limb amputation: 16% v 100%; p<0.05; n=10-21). MicroRNA expression profiling and confirmatory tests indicated that CD34 Exo are significantly enriched with pro-angiogenic miRNAs such as miR126. Treatment with CD34 Exo in mouse hind limb ischemic tissue increased mature miR126 expression, but not endogenous mouse pri-miR126, and activated pro-angiogenic miR126 target-gene expression. Treatment of CD34 Exo lacking miR126 abolished this increase and diminished the pro-angiogenic function of CD34 Exo-treated endothelial cells. This data suggests that miR126 is important for CD34 Exo function. Interestingly, confocal imaging and flow cytometry analyses revealed that CD34 Exo were selectively internalized by endothelial cells when injected to the ischemic tissue. Live imaging using fluorescent miR126 confirmed that CD34 Exo directly transfers miR126 and possibly other yet to be identified moieties from its repertoire specifically to endothelial cells in the ischemic tissue. Conclusion: CD34+ exosomes transfer miR126 to endothelial cells to induce angiogenesis and ischemic tissue repair. The functional benefits associated with CD34+ cell therapy may be mediated by exosomes-mediated targeted transfer of angiogenic microRNAs to endothelial cells.