Abstract 15142: Endogenous Cardiac Stem Cells’ Activation in Response to Injury Potentiates Their Regenerative Ability

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Andrew J Smith ◽  
Iolanda Aquila ◽  
Beverley J Henning ◽  
Mariangela Scalise ◽  
Bernardo Nadal-Ginard ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cardiac Injury ◽  
Fold Increase ◽  
Cardiac Stem Cells ◽  
Post Injury ◽  
Activated State ◽  
Potential Benefits ◽  
Therapeutic Avenue

The identification of resident, endogenous cardiac stem cells (eCSCs) has re-shaped our understanding of cardiac cellular physiology, while offering a significant potential therapeutic avenue. The biology of these cells must be better understood to harness their potential benefits. We used an acute dose (s.c.; 5mgkg-1) of isoproterenol (ISO) to induce diffuse cardiac injury, with associated eCSC activation, in rats. As peak eCSC activation was at 24 hours post ISO-injury, c-kitpos eCSCs were isolated, characterised and their potential for growth and regenerative potential was assessed in vitro and in vivo, respectively. Activated eCSCs showed increased cell cycling activity (51+1% in S- or G2/M phases vs. 9+2% of quiescent), Ki67 expression (56+7% vs. 10+1%) and TERT expression (14-fold increase vs. quiescent). When directly harvested in culture, activated eCSCs showed augmented proliferation, clonogenicity and cardiosphere formation compared to quiescent eCSCs. Activated eCSCs showed increases in expression of numerous growth factors, particularly HGF, IGF-1, TGF-β, periostin, PDGF-AA and VEGF-A. Furthermore, significant alterations were found in the miRnome, notably increased miR-146b and -221, and decreased miR-192 and -351. ISO+5FU was administrated to mice to induce a model of chronic dilated cardiomyopathy, which is characterized by the ablation of eCSCs and the absence of cardiomyocyte replenishment. In these mice with chronic heart failure, freshly isolated quiescent eCSCs or activated eCSCs (2d post-ISO) were injected through the tail vein. 28 days after injection, activated but not quiescent eCSCs re-populated the resident CSC pool, promoted robust new cardiomyocyte formation and improved cardiac function when compared to saline-treated mice. Dual-labelling with BrdU and EdU at selected stages after ISO injury determined that activated eCSCs returned to a quiescent level by 10 weeks post-injury. In conclusion, CSCs rapidly switch from a quiescent to an activated state to match the myocardial needs for myocyte replacement after injury and then spontaneously go back to quiescence. Harnessing the molecules regulating this process may open up future novel approaches for effective myocardial regeneration.

scholarly journals HOPX+ injury-resistant intestinal stem cells drive epithelial recovery after severe intestinal ischemia

2021 ◽  
Author(s):  
Amy Stieler Stewart ◽  
Cecilia Renee Schaaf ◽  
Jennifer A. Luff ◽  
John M. Freund ◽  
Thomas C. Becker ◽  
...  
Keyword(s):  
Stem Cells ◽  
Intestinal Ischemia ◽  
Cellular Proliferation ◽  
Vascular Occlusion ◽  
Epithelial Stem Cells ◽  
Post Injury ◽  
Direct Role ◽  
Activated State

Intestinal ischemia is a life-threatening emergency with mortality rates of 50-80% due to epithelial cell death and resultant barrier loss.Loss of the epithelial barrier occurs in conditions including intestinal volvulus and neonatal necrotizing enterocolitis.Survival depends on effective epithelial repair; crypt-based intestinal epithelial stem cells (ISCs) are the source of epithelial renewal in homeostasis and after injury. Two ISC populations have been described: 1) active ISC [aISC; highly proliferative; leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5+) positive or sex-determining region Y-box 9 -antigen Ki67 positive (SOX9+Ki67+)] and 2) reserve ISC [rISC; less proliferative; homeodomain-only protein X positive (HOPX+)].The contributions of these ISCs have been evaluated both in vivo andin vitrousing a porcine model of mesenteric vascular occlusion to understand mechanisms that modulate ISC recovery responses following ischemic injury. In our previously published work, we observed that rISC conversion to an activated state was associated with decreased HOPXexpression during in vitrorecovery. In the present study, we wished to evaluate the direct role of HOPXon cellular proliferation during recovery after injury. Our data demonstrated that during early in vivo recovery, injury-resistant HOPX+cells maintain quiescence. Subsequent early regeneration within the intestinal crypt occurs around 2 days post injury, a period in which HOPX expression decreased. When HOPX was silenced in vitro,cellular proliferation of injured cells was promoted during recovery. This suggests that HOPXmay serve a functional role in ISC mediated regeneration after injury and could be a target to control ISC proliferation.

Recombinant DEK Enhances Ex Vivo expansion of Human Cord Blood and Mouse Bone Marrow Hematopoietic Stem Cells in a CXCR2- and Hspg-Dependent Manner

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 779-779
Author(s):  
Maegan L. Capitano ◽  
Nirit Mor-Vaknin ◽  
Maureen Legendre ◽  
Scott Cooper ◽  
David Markovitz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Colony Formation ◽  
Ex Vivo ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Inhibitory Function

Abstract DEK is a nuclear DNA-binding protein that has been implicated in the regulation of transcription, chromatin remodeling, and mRNA processing. Endogenous DEK regulates hematopoiesis, as BM from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cell (HSC) engrafting capability (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Moreover, recombinant mouse (rm) DEK inhibits HPC colony formation in vitro. We now show that rmDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers (~2 fold) and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being more sensitive than control mice to this suppression. In contrast, in vivo administration of rmDEK to wild type and DEK-/- mice enhanced numbers of phenotypic LT-HSC. This suggests that DEK may enhance HSC numbers by blocking production of HPCs. We thus assessed effects of DEK on ex vivo expansion of human CD34+ cord blood (CB) and mouse Lin- BM cells stimulated with SCF, Flt3 ligand, and TPO. DEK significantly enhanced ex vivo expansion of rigorously-defined HSC by ~3 fold both on day 4 (~15 fold increase from day 0) and 7 (~29 fold increase from day 0) when compared to cells expanded without DEK. Expanding HSC with DEK also resulted in a decrease in the percentage of apoptotic HSC. Further studies were done to better define how DEK works on HSC and HPC. As extracellular DEK can bind to heparan sulfate proteoglycans (HSPG), become internalized, and then remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 673; Saha et al., 2013, PNAS, 110: 6847), we assessed effects of DEK on the heterochromatin marker H3K9He3 in the nucleus of purified mouse lineage negative, Sca-1 positive, c-Kit positive (LSK) BM cells by imaging flow cytometry. DEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK cells, indicating that rmDEK can be internalized by LSK cells and mediate heterochromatin formation. We also investigated whether inhibiting DEK's ability to bind to HSPG would block the inhibitory function of DEK in HPC. Blocking the synthesis of, the surface expression of, and the binding capability of HSPG blocked the inhibitory effect of DEK on colony formation. Blocking the ability of DEK to bind to HSPG also blocks the expansion of HSC in ex vivo expansion assays, suggesting that DEK mediates its function in both HSC and HPC by binding to HSPG but with opposing effects. To further evaluate the biological role of rmDEK, we utilized single-stranded anti-DEK aptamers that inactivate its function. These aptamers, but not their control, neutralized the inhibitory effect of rmDEK on HPC colony formation. Moreover, treating BM cells in vitro with truncated rmDEK created by incubating DEK with the enzyme DPP4 (DEK has targeted truncation sites for DPP4) eliminated the inhibitory effects of DEK, suggesting that DEK must be in its full- length form in order to perform its function. Upon finding that DEK has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, and as DEK is a chemoattractant for mature white blood cells, we hypothesized that DEK may manifest at least some of its actions through CXCR2, the receptor known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case, we first confirmed expression of CXCR2 on the surface of HSC and HPC and then determined if neutralizing CXCR2 could block DEK's inhibitory function in HPC. BM treated in vitro with rmDEK, rhIL-8, or rmMIP-2 inhibited colony formation; pretreating BM with neutralizing CXCR2 antibodies blocked the inhibitory effect of these proteins. DEK inhibition of CFU-GM colony formation is dependent on Gai-protein-coupled receptor signaling as determined through the use of pertussis toxin, which is a mechanism unique to DEK, as we have previously reported that IL-8 and MIP-1a are insensitive to the inhibitory effects of pertussis toxin. Blocking the ability of DEK to bind to CXCR2 also inhibited the expansion of HSC in an ex vivo expansion assay. This suggests that DEK binds to CXCR2, HSPG or both to mediate its function on HPC and HSC, enhancing HSC but decreasing HPC numbers. Therefore, DEK may be a crucial regulatory determinant of HSC/HPC function and fate decision that is utilized to enhance ex vivo expansion of HSC. Disclosures No relevant conflicts of interest to declare.

scholarly journals A novel microRNA-based strategy to expand the differentiation potency of stem cells

2019 ◽  
Author(s):  
María Salazar-Roa ◽  
Marianna Trakala ◽  
Mónica Álvarez-Fernández ◽  
Fátima Valdés-Mora ◽  
Cuiqing Zhong ◽  
...  
Keyword(s):  
Stem Cells ◽  
De Novo ◽  
Cardiac Injury ◽  
Dna Methyltransferases ◽  
Short Exposure ◽  
Differentiation Potential ◽  
Differentiation Capacity ◽  
Tetraploid Complementation

SUMMARYFull differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) results in expanded differentiation potency as well as improved efficiency in stringent assays such as tetraploid complementation and human-mouse interspecies chimerism. Mechanistically, these effects are mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasing of DNA methylation. As a proof of concept, miR-203 improves differentiation and maturation of PSCs into cardiomyocytes in vitro as well as cardiac regeneration in vivo, after cardiac injury. These data support the use of transient exposure to miR-203 as a general and single method to reset the epigenetic memory in PSCs, and improve their use in regenerative medicine.

Elucidating the role of P63 during development of the mammalian nervous systems

2007 ◽  
Vol 30 (4) ◽  
pp. 79
Author(s):  
Sagar Dugani ◽  
Annie Paquin ◽  
David R. Kaplan ◽  
Freda D. Miller
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Cortical Neurons ◽  
Sympathetic Neurons ◽  
Fold Increase ◽  
P53 Family ◽  
Ongoing Work ◽  
Cortical Precursors

Background: The protein p63, a recently discovered member of the p53 family of proteins, is implicated in the maintenance and differentiation of stem cells in the epidermis and is involved in the regulation of naturally-occurring cell death in sympathetic neurons of the peripheral nervous system. Since initial data from our laboratory indicated that p63 is also widely expressed in stem cells and neurons within the developing brain, we assessed its involvement in regulating the genesis and survival of developing cortical neurons. As neurogenesis is initiated at embryonic day 12 (E12), we isolated cortical precursors from p63-/- embryos at E14 and cultured them for 2 days in vitro (DIV). Methods: Based on immunocytochemistry to known markers of apoptosis and neurons, we assessed the level of cell death and neurogenesis. Results: Compared to p63+/+ cortical precursors, p63-/- precursors from littermates showed a 50 % reduction in neuronal death, as assessed by the apoptosis marker, cleaved caspase 3. Interestingly, the proportion of neurons and astrocyte precursors, the latter identified by S100b was also reduced in p63-/- embryos, as compared to p63+/+ littermates. Conclusions: These results suggest that p63 may be involved in the regulation of cell survival and in the differentiation of precursors into neurons and astrocytes. To assess the former, we overexpressed TAp63a, a full-length isoform of p63, in E12/13 cortical precursors and assessed the level of cell death after 2 DIV. Compared to control cells, cells transfected with TAp63a demonstrated a 2-fold increase in cell death. Ongoing work will characterize p63 involvement in differentiation of precursor cells into neurons and astrocytes. To assess if these findings are relevant in vivo, we will use p63flox,flox X Nextin-Cre mice, which have p63 specifically ablated in neural precursors. We will analyze the survival, proliferation, and fate of these p63-/- cells. Together, these studies will help to determine a role for p63 in neural proliferation and apoptosis, processes central to development and response to injury.

Abstract 22: Identification of the Nature of Cardiac Resident c-kit + Cells

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Chen-Leng Cai ◽  
Nishat Sultana ◽  
Lu Zhang ◽  
Jianyun Yan ◽  
Jiqiu Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Receptor Tyrosine Kinase ◽  
Cardiac Injury ◽  
Cardiac Stem Cells ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Heart Repair ◽  
Bona Fide ◽  
Myocardial Marker

Identifying a bona fide population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. For more than a decade, c-kit, a receptor tyrosine kinase expressed in certain types of hematopoietic stem cells, has been recognized as a marker of resident CSCs in mammals. It was shown that c-kit + cells are multipotent, with differentiation potential to become cardiomyocytes, endothelial, and smooth muscle cells in vitro and after cardiac injury. Here, we provide new insights into the nature of cardiac resident c-kit + cells. By targeting the c-kit locus with several reporter genes in mice, we unexpectedly found that c-kit + cells rarely co-localizes with cardiac progenitor marker Nkx2.5 or myocardial marker cTnT. Instead, c-kit labels an endocardial population from embryonic stage to adulthood. After acute cardiac injury, the c-kit + cells still retain their endothelial identity and do not become cardiomyocytes. Our study supports the notion that cardiac c-kit + cells are in fact endothelial cells and not CSCs. This finding suggests an urgent need to re-evaluate the mechanisms by which c-kit + cells contribute to heart repair or regeneration given their endothelial identity.

Enriched MicroRNA-126 Bioactivity Marks the Primitive Compartment In AML and Regulates LSC Numbers

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 94-94
Author(s):  
Eric Lechman ◽  
Bernhard Gentner ◽  
Hidefumi Hiramatsu ◽  
Kristin J Hope ◽  
Katsuto Takenaka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Colony Formation ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Biologically Active ◽  
Reporter System ◽  
Differentiation Marker

Abstract Abstract 94 Previous work has shown miRNAs to be dysregulated in acute myeloid leukemia (AML), however, there is little known regarding miRNA expression and function in leukemia stem cells (LSC). In order to elucidate the role of miRNA in LSC, we performed miRNA profiling on fractionated subpopulations of primary AML patient samples. Supervised analysis guided by the in vivo SCID leukemia initiating capacity (SL-IC) of each sub-population generated a unique miRNA signature associated with LSC enriched fractions. An in vitro antagomir-based functional miRNA knockdown screen identified miR-126, our top array candidate, for further study. After RT-PCR validation, the biological activity of miR-126 was confirmed at single cell resolution by using a novel bidirectional lentivirus miRNA reporter system in the 8227 cell line in vitro and within primary AML patient samples xenografted into immune-deficient NSG mice. These data suggest that primitive AML cells may express high levels of bioactive miR-126 relative to more “differentiated” blast populations. To test the hypothesis that AML stem cells are marked by high miR-126 bioactivity, we FACS sorted miR-126 genetic reporter vector transduced primary AML patient samples and transplanted these populations into immune-compromised secondary mouse recipients. The results of these proof-of-concept experiments demonstrates our ability to prospectively isolate LSC enriched fractions in all 4 AML patient samples tested using only a single biomarker, miR-126. Finally, to understand the functional relevance of miR-126 expression within primitive AML cells, stable enforced expression and knockdown of miR-126 was achieved using lentiviral vectors. Enforced expression of miR-126 in CD34+CD38- 8227 cells resulted in reduced AML blast colony formation, an increase/maintenance of CD34+ cells and a decrease in differentiation marker positive (CD14, CD15) AML blasts. Similarly, enforced miR-126 expression in 4 primary AML xenografts resulted in a several fold increase of CD34+CD117+ lentivirus marked leukemia cells after 12 weeks. In addition, the miR-126 cells showed reduced differentiation marker expression (CD14, CD15) with no significant differences in AML graft size. To determine if the expanded population had SL-IC activity or was a downstream leukemic progenitor, limiting dilution assays were performed by transplantation of FACS sorted lentivirus marked cells into secondary recipient mice for 12 weeks. A 13 fold increase in LSC activity was observed with miR-126 forced expression compared to control vector expressing cells. These data suggest that high levels of miR-126 bioactivity may support self-renewal/maintenance of primitive AML cells at the cost of aberrant differentiation. Conversely, in vitro knockdown of miR-126 in CD34+CD38- 8227 cells increased AML blast colony formation, while no phenotype was observed in xenotransplanted primary AML, with secondary LDA transplant experiments ongoing. Target prediction algorithms and previously described target genes were used to ascertain the principal signaling pathway(s) under direct control of miR-126 in primitive AML cells. In summary, these experiments demonstrate that miR-126 is more abundantly expressed and biologically active within the leukemia stem/progenitor cell compartment of the AML functional hierarchy and serves to regulate AML stem cell numbers. Disclosures: No relevant conflicts of interest to declare.

scholarly journals N-arachidonoyl-L-serine (AraS) Possesses Proneurogenic Properties in Vitro and in Vivo after Traumatic Brain Injury

2013 ◽  
Vol 33 (8) ◽  
pp. 1242-1250 ◽  
Author(s):  
Ayelet Cohen-Yeshurun ◽  
Dafna Willner ◽  
Victoria Trembovler ◽  
Alexander Alexandrovich ◽  
Raphael Mechoulam ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Cannabinoid Receptor ◽  
Fold Increase ◽  
Lesion Volume ◽  
Post Injury ◽  
Neuroprotective Effects ◽  
Neurobehavioral Function

N-arachidonoyl-L-serine (AraS) is a novel neuroprotective endocannabinoid. We aimed to test the effects of exogenous AraS on neurogenesis after traumatic brain injury (TBI). The effects of AraS on neural progenitor cells (NPC) proliferation, survival, and differentiation were examined in vitro. Next, mice underwent TBI and were treated with AraS or vehicle. Lesion volumes and clinical outcome were evaluated and the effects on neurogenesis were tested using immunohistochemistry. Treatment with AraS led to a dose-dependent increase in neurosphere size without affecting cell survival. These effects were partially reversed by CB1, CB2, or TRPV1 antagonists. AraS significantly reduced the differentiation of NPC in vitro to astrocytes or neurons and led to a 2.5-fold increase in expression of the NPC marker nestin. Similar effects were observed in vivo in mice treated with AraS 7 days after TBI. These effects were accompanied by a reduction in lesion volume and an improvement in neurobehavioral function compared with controls. AraS increases proliferation of NPCs in vitro in cannabinoid-receptor-mediated mechanisms and maintains NPC in an undifferentiated state in vitro and in vivo. Moreover, although given at 7 days post injury, these effects are associated with significant neuroprotective effects leading to an improvement in neurobehavioral functions.

scholarly journals Estrogen is required for maintaining the quality of cardiac stem cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245166
Author(s):  
Al Shaimaa Hasan ◽  
Lan Luo ◽  
Satoko Baba ◽  
Tao-Sheng Li
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Potential Role ◽  
Telomerase Activity ◽  
Estrogen Deficiency ◽  
Cardiac Stem Cells

Compared to the age-matched men, the incidence of cardiovascular diseases is lower in premenopausal but higher in postmenopausal women, suggesting the cardio-protective role of estrogen in females. Although cardiac stem cells (CSCs) express estrogen receptors, yet the effects of estrogen on CSCs remain unclear. In this study, we investigated the potential role of estrogen in maintaining the quality of CSCs by in vivo and in vitro experiments. For the in vivo study, estrogen deficiency was induced by ovariectomy in 6-weeks-old C57BL/6 female mice, and then randomly given 17β-estradiol (E2) replacements at a low dose (0.01 mg/60 days) and high dose (0.18 mg/60 days), or vehicle treatment. All mice were killed 2 months after treatments, and heart tissues were collected for ex vivo expansion of CSCs. Compared to age-matched healthy controls, estrogen deficiency slightly decreased the yield of CSCs with significantly lower telomerase activity and more DNA damage. Interestingly, E2 replacements at low and high doses significantly increased the yield of CSCs and reversed the quality impairment of CSCs following estrogen deficiency. For the in vitro study, twice-passaged CSCs from the hearts of adult healthy female mice were cultured with the supplement of 0.01, 0.1, and 1 μM E2 in the medium for 3 days. We found that E2 supplement increased c-kit expression, increased proliferative activity, improved telomerase activity, and reduced DNA damage of CSCs in a dose-dependent manner. Our data suggested the potential role of estrogen in maintaining the quality of CSCs, providing new insight into the cardio-protective effects of estrogen.

scholarly journals Abnormal Cytokine Production By Mast Cell Cultures from Sickle Cell Anemia Patients in Response to Inflammatory Stimuli and to Co-Culture with Eosinophils

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3566-3566
Author(s):  
Myriam Salazar-Terreros ◽  
Kleber Yotsumoto Fertrin ◽  
Nicolas Moreno Reyes ◽  
Fernando Ferreira Costa ◽  
Carla F. Franco-Penteado
Keyword(s):  
Mast Cell ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Peripheral Blood ◽  
Cytokine Production ◽  
Fold Increase ◽  
Individual Response ◽  
Activated State

Mast cell function (MC) in pathologic states can be studied through their ability to secrete mediators in vitro depending on MC phenotype and the nature of the stimuli. Reports on MC mediators in sickle cell anemia (SCA) patients are scarce, but clinical signs of MC activation syndrome, such as increased plasma histamine in vaso‐occlusive crisis (VOC), and normal or slightly elevated serum tryptase have been reported. However, assessing the biological relevance of MC as a cytokine source is more challenging because it is unclear under which circumstances they secrete those products in vivo, or if the cytokines measured systemically stem from a different cell type. We aimed to investigate the profile of mediators involved in the inflammatory process produced by MC in SCA. Methods:The supernatant of 5-week old MC cultures (17 SCA, 8 HV) obtained from peripheral blood CD34+ cells from 29 SCA patients and 13 healthy volunteers (HV) was analyzed using a multiplex platform and colorimetric assays for endothelin-1 (ET-1) and substance P (SP) (10 SCA, 6 HV). A correlation matrix (Pearson correlations, R software, v. 3.6.1) was generated using laboratory and clinical data chosen based on their value as inflammatory or prognosis markers (hydroxyurea [HU] treatment, fetal hemoglobin [HbF], hemoglobin [Hb], vaso-occlusive crisis [VOC], percentage of peripheral blood neutrophils, eosinophils (Eos), basophils, erythroblasts, and reticulocytes), MC surface expression of CD117, CD48 and CD63, and the supernatant content of 11 cytokines. To investigate MC cytokine release, we tested the supernatants from Eos-MC co-cultures (3:1 ratio), and after stimulation with ET-1 (20 nM), SP (10 µM) and imatinib (20 µg/ml)(n=3 per treatment). Results: Out of 26 cytokines, we found elevated levels of the following in the supernatants of SCA-MC cultures (data represented as mean in pg/ml±SE): TNFα: SCA=88.7±18.4, HV=32.6±3.8; IFNγ: SCA=55.3±11.2, HV=15.7±1.8; MCP1: SCA=555.0±147.2, HV=145.3±35.2; RANTES: SCA=24.7±3.9, HV=10.7±1.8 (p<0.05). However, SCA-MC from patients treated with HU (n = 11) showed higher values of IL-1b, IL-4, IL-5, IL-9, IL-15, and FGF than HV (n=8) and HU-free patients (n=6) (p<0.05). Supernatants from SCA-MC had higher ET-1 production compared to HV-MC (SCA=16.3±1.2, HV=11.93±1.3, pg/ml, p=0.02) but SP production was similar (SCA=27.9±1.3; HV=31.49±0.7 pg/ml). ET-1 stimulation of MC cultures caused 2-fold increase in IL-1AR production on HV-MC, but failed to produce any effect on SCA-MC. Similarly, imatinib reduced FGF only in HV-MC samples (HV: 15.1±3.5, HV-HU: 4.0±1.6, pg/ml). No effect on cytokine production was observed with SP. Conversely, Eos-MC cocultures showed a 10- and 4-fold increase of IL-5 and IL-9, respectively, regardless of the origin of Eos (HV or SCA). SCA-MC/SCA-Eos co-cultures had elevated proinflammatory (IL-1b, IL-12, TNF-α) and angiogenic (FGF, VEGF) cytokines, RANTES, IL-7, IL-4, and IL1-RA compared with SCA-MC/HV-Eos and HV-MC/HV-Eos (p<0.05). Preliminary multiparametric analysis on data from SCA patients showed a strong negative correlation between HU therapy and VGEF production, and between HbF levels and CD63 expression (MC activation marker).We also found a positive correlation between history of VOC and eotaxin-1 produced by SCA-MC. Conclusions: We found that MC responses depend both on the origin of the cultured cell and the stimuli utilized. Despite differences between in vitro and in vivo MC populations, our data show that cultured SCA-MC have a sustained activated state and produce a repertoire of mediators that could contribute to a perivascular microenvironment in favor of leukocyte and endothelium activation. In terms of cytokine production, cultured SCA-MC were more sensitive to stimulation by SCA-Eos than by HV-Eos, which may be relevant to the pathophysiology of airway inflammation in SCA patients with asthma. Differences in cytokine production between SCA-MC cultures from patients treated or not with HU may reflect the variability in adherence to treatment, individual response to each compound, or epigenetic modifications during the MC differentiation process that affect the phenotype of the mature MC. These results support that mediators produced by MC can contribute to the chronic inflammatory state and may be implicated in exacerbated responses to eosinophil activation in SCA. Disclosures Fertrin: Agios Pharmaceuticals, Inc.: Research Funding.

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