Spry1 Plays Selective Hematopoietic Roles as An Erythropoietin - Modulated Positive Regulator of Erythropoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 777-777
Author(s):  
Pradeep Sathyanarayana ◽  
Anamika Pradeep ◽  
Jonathan D. Licht ◽  
Don M. Wojchowski
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Cell Formation ◽  
Strong Increase ◽  
Erythroid Progenitors ◽  
Kinase Activation ◽  
Cellular Models ◽  
Erythropoietin Treatment ◽  
Molecular Adaptors

Abstract Abstract 777 The four vertebrate Sprouty (Spry1-4) proteins are molecular adaptors, best known as negative regulators of MAP kinase activation mediated by FGFR, VEGFR and RET. Prior studies in human hematopietic stem cells and zebrafish implicated Spry proteins in stem cell development. Presently, we have ascertained the role of Spry1 in erythroid development using cellular models and in knockout mice. Treatment of UT7 erythropoietin-responsive cells led a strong increase in phosphorylation of Spry1 and Spry2 on critical N-terminal tyrosine sites of these proteins (Y53 and Y55, respectively). UT7 cells engineered to constitutively express Spry1 also demonstrated decreased ERK activation in response to erythropoietin treatment. Spry expression was measured in developing primary bone marrow (pro)erythroblasts by real time PCR. Spry1 was expressed most prominently in erythroblasts at a level 40 times higher than Spry 2-4. Furthermore, Spry1 expression rose markedly as erythroblasts matured. To determine the role of Spry1 in murine hematopoiesis, conditional, LoxP flanked allele of Spry1 was crossed with Mx1-Cre transgenic mice and Spry1 was deleted in murine marrow by injection of mice with poly pIpC. Efficient deletion of the Spry1 gene in murine marrow did not affect lymphocytes or granulocytes and selectively led to an increased reticulocyte count (8.9% +/- 0.2% in Spry1 deleted vs. 4.9 +/- 0.5% in control mice, p<0.002). Deletion of Spry1 led to activation of splenic erythropoiesis with a four fold enrichment of CD71high, Ter119pos precursors in Mx1-Cre; Spry1flox/flox animals compared to Mx1-Cre;Spry1flox/+ animals. In ex vivo expansion cultures, however, erythroid progenitors, were significantly compromised in their intrinsic capacity to form KitnegCD71highTer119neg and KitnegCD71highTer119pos erythroblasts. Collectively these data suggest that during hematopoiesis, SPRY1 acts selectively as a non-redundant novel positive effector of EPO- dependent red cell formation. Disclosures: No relevant conflicts of interest to declare.

Conditioned Medium Represents a Useful Solution to Increase the Expansion of Multipotent Progenitors with Strong Platelet Engraftment Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4276-4276
Author(s):  
Ahmad Abu-Khader ◽  
Gwendoline Bugnot ◽  
Manal Alsheikh ◽  
Roya Pasha ◽  
Nicolas Pineault
Keyword(s):  
Cell Expansion ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Background Level ◽  
Erythroid Progenitors ◽  
Cellular Mechanisms ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Cd34 Cells

Abstract Delayed neutrophil and platelet engraftment is a significant issue of cord blood (CB) transplantation. Ex vivo expansion of CB hematopoietic stem and progenitor cells (HSPC) before infusion has been shown to accelerate hematopoietic recovery in patients. Recently, we reported that serum free medium (SFM) conditioned with osteoblasts derived from human bone marrow (BM) mesenchymal stromal cells, referred as M-OST CM, was superior to SFM or MSC CM for the expansion of CB CD34+ cells, and that HSPC expanded in M-OST CM provided better platelet engraftment. Since large number of expanded cells were transplanted in the original study, it was not possible to estimate the increased expansion of HSPC with short-term (ST) and long-term (LT) thrombopoietic and BM engraftment activities. The objectives herein were to investigate these shortcomings using limit dilution analysis (LDA) in transplantation assay and to investigate the cellular mechanisms at play. M-OST CM was prepared by conditioning SFM with immature M-OST overnight. CB CD34+ cells were expanded in M-OST CM or in SFM (defined as control) for 7 days with SCF, FL and TPO. CB cell expansion was significantly greater in M-OST CM cultures vs. SFM control (2.4 ±0.9 fold, mean ± SD, n=4, p=0.01). LDA transplantation assays were done by infusing the progeny of 500-8000 CD34+ cells in NSG mice. First, we compared the ST (< 31 days) and LT (˃ 100 days) thrombopoietic activities of expanded HSPC by measuring circulating human platelets (hPLT). The threshold for hPLT engraftment was set above the mean background level measured in control mice + 1SD. The median ST levels of hPLT in M-OST mice tended to be greater (2.5-fold, p˃0.05) in M-OST recipients (21 mice/condition, n=2). The frequency of ST hPLT HSPC estimated by LDA was 3.4 ±0.2 fold higher in M-OST CM cultures though the difference vs. control was not significant (p=0.11). LT hPLT levels were significantly greater in M-OST recipients (median 33 vs. 8 hPLT/uL blood, p=0.0027). Consistent with this, the frequency of HSPC with LT hPLT engraftment was increased in M-OST CM cultures (3.5±0.8 fold, p<0.04). Considering the differences in cell expansion, the net expansion of HSPC with ST and LT hPLT engraftment were raised by 5.5 ±1.7 and 6.0 ±3.4 fold in M-OST CM cultures vs. control (n=2). Next, LT human BM engraftment was analyzed at week 16. Preliminary results (13 mice/condition) suggest that the frequency of LT Scid repopulating cells (SRC) was increased by 27% in the M-OST CM culture vs. SFM control (frequency of 1/2878 vs. 1/3626 of day 0 starting cell). Next, we set to determine how M-OST CM increases the thrombopoietic activity of expanded CB HSPC. First, cytometry analysis (CD34, CD38, CD45RA, CD90 and CD123) revealed that M-OST CM preferentially increased the expansion of common myeloid progenitors (CMP, 8-fold, p=0.2, n=3), megakaryocyte-erythroid progenitors (MEP, 7-fold, p=0.02) and granulocyte-macrophage progenitors (GMP, 9-fold, p=0.02) vs. SFM control. Expansion of HSC-enriched cells was unchanged while that of multipotent progenitors (MPP) was reduced 2-fold (p<0.05). We set to confirm these results by culturing purified primary CB HSPC subsets in M-OST CM or SFM; M-OST CM induced greater expansions of MEP (3-fold), GMP (˃10-fold) while expansion in MPP cultures was greater with SFM control (1.5-fold). No growth was noted with the HSC and CMP cultures likely due to low sort yields. To complement these findings, we measured the expansion of myeloid CFU progenitors and long term culture-initiating cells (LTC-IC) by LDA. The total number of CFU was increased 2.4-fold (<0.02, n=4) by M-OST CM due mostly to increased expansion of CFU-G/GM colonies (2-fold, p<0.05) and BFU-E (2-fold, p=0.05). M-OST CM also sustained a 3.4-fold increase in LTC-IC expansion vs. SFM culture, though this finding remains to be confirmed in ongoing experiments. Finally, we investigated the effect of M-OST CM on the chemotaxis of HSPC toward SDF-1 since we previously reported increased expression of its receptor CXCR4 on CB cells in M-OST CM cultures. M-OST CM HSPC showed a modest 15% increase in migration vs. SFM control (n=4, p=0.10). In conclusion, our results demonstrate that the ST and LT hPLT engraftment activities of ex vivo expanded CB HSPC can be increased 5-6 folds by the use of M-OST CM due to the expansion of immature CB HSPC subsets including perhaps LT SRC. Whether M-OST CM can also modulate the homing activity of HSPC remains unclear. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional Requirements of a Samd14-Capping Protein Interaction in Stress Erythropoiesis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 288-288
Author(s):  
Suhita Ray ◽  
Linda Chee ◽  
Nicholas T. Woods ◽  
Kyle J Hewitt
Keyword(s):  
Steady State ◽  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Interacting Proteins ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Capping Protein ◽  
Sam Domain ◽  
Stress Erythropoiesis

Abstract Stress erythropoiesis describes the process of accelerating red blood cell (RBC) production in anemia. Among a number of important mediators of stress erythropoiesis, paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the activation/function of stress erythroid progenitors. Whereas many critical factors required to drive erythropoiesis in normal physiological conditions have been described, whether distinct mechanisms control developmental, steady-state, and stress erythropoiesis in anemia is poorly understood. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene is transcriptionally upregulated in a model of acute hemolytic anemia induced by the RBC-lysing chemical phenylhydrazine. Samd14 is regulated by GATA binding transcription factors via an intronic enhancer (Samd14-Enh). In a mouse knockout of Samd14-Enh (Samd14-Enh -/-), we established that the Samd14-Enh is dispensable for steady-state erythropoiesis but is required for recovery from severe hemolytic anemia. Samd14 promotes c-Kit signaling in vivo and ex vivo, and the SAM domain of Samd14 facilitates c-Kit-mediated cellular signaling and stress progenitor activity. In addition, the Samd14 SAM domain is functionally distinct from closely related SAM domains, which demonstrates a unique role for this SAM domain in stress signaling and cell survival. In our working model, Samd14-Enh is part of an ensemble of anemia-responsive enhancers which promote stress erythroid progenitor activity. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. To identify potential Samd14-interacting proteins that mediate its function, we performed immunoprecipitation-mass spectrometry on the Samd14 protein. We found that Samd14 interacted with α- and β heterodimers of the F-actin capping protein (CP) complex independent of the SAM domain. CP binds to actin during filament assembly/disassembly and plays a role in cell morphology, migration, and signaling. Deleting a 17 amino acid sequence near the N-terminus of Samd14 disrupted the Samd14-CP interaction. However, mutating the canonical RxR of the CP interaction (CPI) motif, which is required for CP-binding in other proteins, does not abrogate the Samd14-CP interaction. Moreover, replacing this sequence with the canonical CPI domain of CKIP-1 completely disrupts the interaction, indicating that other sequence features are required to maintain the Samd14-CP complex. Ex vivo knockdown of the β-subunit of CP (CPβ), which disrupts the integrity of the CP complex, decreased the percentage of early erythroid precursors (p&lt;0.0001) and decreased (3-fold) progenitor activity as measured by colony formation assays (similar to knockdown of Samd14). Taken together, these data indicate that Samd14 interacts with CP via a unique CP binding (CPB) domain, and that the CP complex coordinates erythroid differentiation in stress erythroid progenitors. To test the function of the Samd14-CP complex, we designed an ex vivo genetic complementation assay to express Samd14 lacking the CPB-domain (Samd14∆CPB) in stress erythroid progenitors isolated from anemic Samd14-Enh -/- mice. Phospho-AKT (Ser473) and phospho-ERK (Thr202/Tyr204) levels in Samd14∆CPB were, respectively, 2.2 fold (p=0.007) and ~7 fold (n=3) lower than wild type Samd14 expressing cells, 5 min post SCF stimulation. Relative to Samd14, Samd14∆CPB expression reduced burst forming unit-erythroid (BFU-E) (2.0 fold) and colony forming unit-erythroid (CFU-E) (1.5 fold). These results revealed that the Samd14-CP interaction is a determinant of BFU-E and CFU-E progenitor cell levels and function. Remarkably, as the requirement of the CPB domain in BFU-E and CFU-E progenitors is distinct from the Samd14-SAM domain (which promotes BFU-E but not CFU-E), the function of Samd14 in these two cell types may differ. Ongoing studies will examine whether the function of Samd14 extends beyond SCF/c-Kit signaling and establish cell type-dependent functions of Samd14 and Samd14-interacting proteins. Given the critical importance of c-Kit signaling in hematopoiesis, the role of Samd14 in mediating pathway activation, and our discovery implicating the capping protein complex in erythropoiesis, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional properties of edible insects: a systematic review

2021 ◽  
pp. 1-54
Author(s):  
V. D’Antonio ◽  
N. Battista ◽  
G. Sacchetti ◽  
C. Di Mattia ◽  
M. Serafini
Keyword(s):  
Glucose Metabolism ◽  
Functional Role ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
Edible Insects ◽  
Cellular Models ◽  
Pubmed Database

Abstract Consumption of edible insects has been widely suggested as an environmentally sustainable substitute for meat to reduce GHG emissions. However, the novel research field for edible insects rely on the content of bioactive ingredients and on the ability to induce a functional effect in humans. The goal of this manuscript was to review the available body of evidence on the properties of edible insects in modulating oxidative and inflammatory stress, platelet aggregation, lipid and glucose metabolism and weight control. A search for literature investigating the functional role of edible insects was carried out in the PUBMED database using specific keywords. A total of 55 studies, meeting inclusion criteria after screening, were divided on the basis of the experimental approach: in vitro studies, cellular models/ex vivo studies or in vivo studies. In the majority of the studies, insects demonstrated the ability to reduce oxidative stress, modulate antioxidant status, restore the impaired activity of antioxidant enzymes and reduce markers of oxidative damage. Edible insects displayed anti-inflammatory activity reducing cytokines and modulating specific transcription factors. Results from animal studies suggest that edible insects can modulate lipid and glucose metabolism. The limited number of studies focused on the assessment of anticoagulation activity of edible insects make it difficult to draw conclusions. More evidence from dietary intervention studies in humans is needed to support the promising evidence from in vitro and animal models about the functional role of edible insects consumption.

scholarly journals Squalene: More than a Step toward Sterols

Antioxidants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 688 ◽  
Author(s):  
Marco Micera ◽  
Alfonso Botto ◽  
Federica Geddo ◽  
Susanna Antoniotti ◽  
Cinzia Margherita Bertea ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Free Radicals ◽  
Cardiovascular Diseases ◽  
Defense Mechanisms ◽  
Current Knowledge ◽  
Ex Vivo ◽  
Antioxidant Properties ◽  
Metabolic Intermediate ◽  
Cellular Models

Squalene (SQ) is a natural triterpene widely distributed in nature. It is a metabolic intermediate of the sterol biosynthetic pathway and represents a possible target in different metabolic and oxidative stress-related disorders. Growing interest has been focused on SQ’s antioxidant properties, derived from its chemical structure. Strong evidence provided by ex vivo models underline its scavenging activity towards free radicals, whereas only a few studies have highlighted its effect in cellular models of oxidative stress. Given the role of unbalanced free radicals in both the onset and progression of several cardiovascular diseases, an in depth evaluation of SQ’s contribution to antioxidant defense mechanisms could represent a strategic approach in dealing with these pathological conditions. At present experimental results overall show a double-edged sword role of squalene in cardiovascular diseases and its function has to be better elucidated in order to establish intervention lines focused on its features. This review aims to summarize current knowledge about endogenous and exogenous sources of SQ and to point out the controversial role of SQ in cardiovascular physiology.

scholarly journals Zebrafish Kit ligands cooperate with erythropoietin to promote erythroid cell expansion

2020 ◽  
Vol 4 (23) ◽  
pp. 5915-5924
Author(s):  
Jana Oltova ◽  
Ondrej Svoboda ◽  
Olga Machonova ◽  
Petra Svatonova ◽  
David Traver ◽  
...  
Keyword(s):  
Suspension Culture ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Erythroid Cell ◽  
Loss Of Function ◽  
Erythroid Progenitors ◽  
Evolutionarily Conserved

Abstract Kit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.

The Role of Microtubule Regulatory Protein Stathmin (STMN1) in Megakaryopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4591-4591
Author(s):  
Camelia Iancu-Rubin ◽  
Joseph Tripodi ◽  
Vesna Najfeld ◽  
George F. Atweh
Keyword(s):  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Critical Role ◽  
Regulatory Protein ◽  
Active Form ◽  
Fish Analysis ◽  
Ploidy Levels ◽  
Significant Difference

Abstract Abstract 4591 Megakaryopoiesis is a complex process in which hematopoietic progenitor cells proliferate and acquire megakaryocyte (MK)-specific markers then undergo polyploidization (i.e. acquisition of DNA content >2n) and cytoplasmic maturation, and start producing platelets. Polyploidization and platelet formation are highly dependent on microtubule (MT) function. To become polyploid, MK undergo abortive mitosis that is mediated by a mitotic spindle that consists of MT. Mature polyploid MK extend cytoplasmic extensions (i.e. proplatelets) into the vascular space and release platelets into the circulation. MT provide the structural scaffold for the proplatelets and mediate the transport of organelles and specific granules into nascent platelets. Despite the critical role of MT in MK biology, the regulation of MT in MK is poorly defined. Stathmin (STMN1) is a cytosolic phosphoprotein whose major function is to regulate MT function by promoting their depolymerization. We had previously shown that STMN1 is expressed at high levels early during megakaryopoiesis and is downregulated later during MK maturation. We also showed that inhibition of STMN1 expression increased ploidy while its overexpression decreased ploidy of MK-like cell lines. Thus, we hypothesized that the dynamic regulation of STMN1 expression may be necessary for megakaryopoiesis and that perturbing its expression may impair MK polyploidization and platelet production. To test this hypothesis, we developed feline immunodeficiency virus (FIV)-based lentiviruses that express STMN1 to investigate the effects of overexpression in primary MK. Since the depolymerizing activity of STMN1 can be inactivated by a variety of cellular kinases, we generated a STMN1 vectors that expresses wild-type (WT) and another that expresses a contitutively active phosphorylation-deficient mutant of STMN1 (MT). We also developed a vector that expresses GFP as a negative control. Human MK generated ex vivo in liquid culture from CD34+ cells were infected with these different lentiviruses. After ectopic STMN1 expression by RT-PCR and flow cytometry was confirmed, MK differentiation was assessed in the presence or absence of STMN1 overexpression. Uninfected MK and MK infected with GFP lentiviruses differentiated and matured into large, easily recognizable cells with typical nuclear morphology and expressed similar levels of CD41 and CD42b by flow cytometry. The numbers of MK generated in the presence of WT-STMN1 expressing lentiviruses was similar to that generated in the cultures infected with control lentiviruses, while the number of MK generated in the presence of phosphorylation-deficient MT-STMN1 was drasticaly reduced. Similarly, the numbers of CD41+ and CD42b+ MK generated in the presence of MT-STMN1 was reduced two and three times, respectively, suggesting that overexpression of a contitutively active form of STMN1 prevents MK differentiation and maturation. We then evaluated the effects of STMN1 overexpression on MK polyploidization by determining the number X and Y chromosomes by FISH analysis. While a normal diploid cell has one copy of each chromosome, cells with ploidy levels of 4N, 8N and 16N will have 2, 4 and 8 copies, respectively. There was no significant difference between the fraction of polyploid MK infected with control-GFP and those infected with WT-STMN1 lentiviruses. In contrast, the fraction of polyploid MK infected with MT-STMN1 lentiviruses was reduced by approximately 50%, suggesting that STMN1 overexpression impairs the ability of MK to become polyploid. In conclusion, we demonstrated that perturbing the normal downregulation of STMN1 in primary human MK impairs differentiation and polyploidization. Since STMN1 is expressesd at extremely high levels in a variety of human leukemias, we have started assessing STMN1 expression expression in patients with hematological malignancies characterized by striking abnormalities in their MK lineage. Such studies might validate the role of MT regulation in MK biology in vivo and support the development of potential therapeutic strategies to target MT and/or STMN1 function in MK and platelet disorders. Disclosures: No relevant conflicts of interest to declare.

the γ Isoform of the Glucocorticoid Receptor Is Ontogenetically Activated and Predicts Poor Ex-Vivo Expansion of Erythroid Cells From Adult Blood.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 642-642
Author(s):  
Giovanni Migliaccio ◽  
Massimo Sanchez ◽  
Francesca Masiello ◽  
Valentina Tirelli ◽  
Lilian Varricchio ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Optimal Growth ◽  
Human Albumin ◽  
Red Cells ◽  
Erythroid Cells ◽  
Erythroid Progenitors

Abstract Abstract 642 Ex-vivo generated erythroblasts (EBs) represent alternative transfusion products. Adult blood (AB) contains numbers of progenitor cells comparable to those present in cord blood (CB) (106 vs 1.8×106 CD34pos cells in average AB and CB donations) but generates lower numbers of erythroblasts (EBs) (∼4.8×108 vs 6.6×1010, respectively) and, in spite of its numerous advantages, is not considered suitable for ex-vivo EB production. To assess the potential of AB to generate EBs ex-vivo, the growth factors [stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO)] and optimal concentration and addition schedule of dexamethasone (DXM) and estradiol (ES) sustaining maximal EB amplification from AB mononuclear cells (MNC) were defined using media with serum previously defined as human erythroid massive amplification culture (HEMAser). Adult MNC stimulated with SCF and IL-3 in combination with EPO generated low numbers (fold increase ∼2) of EBs at all stages of maturation. Concentration response studies conducted on MNC from 10 different donors, indicated that the further addition to the cultures of DXM and ES (both at 10-6 M) increased (∼6-12-fold) the numbers of EBs generated. Delayed addition and withdrawal experiments indicated that DXM and ES exerted partially overlapping but non-redundant functions. DXM was indispensable to achieve maximal amplification in the first 10 days of culture while ES was required from day 10 on. To determine if variability in glucocorticoid receptor (GR) expression might affect ex vivo generation of EBs, expression of αa and γ GR isoforms (αaGR and γGR) by EBs from 10 AB and 5 CB was investigated. While EBs from all donors expressed αaGR, γGR was not expressed by EBs obtained from CB and from AB that generated high numbers of EBs ex vivo, suggesting that activation of γGR in EBs is ontogenetically activated in a subset of AB and may predicts poor expansion. Ex vivo produced EBs are megaloblastic (30 to 50 μm). EPO decreased their size from 40.1±1.4 to 11.6±0.3 μm by 96 h (p<0.01). Although still macrocytic (adult normocytic red cells are 8 μm), these cells are smaller than fetal red cells (12.5 μm) and therefore suitable for clinical use. Inclusion of bovine components in HEMAser precludes its use for clinical purposes. Therefore, optimal growth factor and hormone combinations identified in HEMAser were used to formulate a medium composed of pharmaceutical grade human albumin, human albumin-based-lipid liposomes and iron-saturated recombinant human-tranferrin (HEMAdef). HEMAdef sustained EB amplification as efficiently as HEMAser from CB MNC and 10-fold higher than HEMAser from AB MNC. Moreover, the numbers of EBs generated in HEMAdef by adult MNC were similar to those generated by CB MNC (750×106 vs 500×106 per 106 MNC from AB and CB, respectively). Assuming that MNC contain 102-103 EB progenitors (CD34pos cells represent 0.1% of MNC and erythroid progenitors represent 10% of CD34pos cells), it was calculated that the generation of 750×106 EBs from the progenitors present in 106 adult MNC required 19-23 divisions, a number below the theoretical Hayflick's limit for somatic cell divisions of 35. These results indicate that at least a subset of AB donors is suitable to produce ex-vivo erythroid cells for transfusion and that it should be possible, by optimizing HEMAdef components, to further increase the number of EBs that can be generated ex-vivo from AB. Disclosures: No relevant conflicts of interest to declare.

Early Erythroid Development Is Enhanced with Hypoxia and Terminal Maturation with Normoxia in a 3D Ex Vivo Physiologic Eythropoiesis Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2453-2453
Author(s):  
Susana Brito dos Santos ◽  
Mark C. Allenby ◽  
Athanasios Mantalaris ◽  
Nicki Panoskaltsis
Keyword(s):  
Mononuclear Cells ◽  
Cytokine Production ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Erythroid Progenitors ◽  
Variable Intensity ◽  
Erythroid Progenitor ◽  
Tnf Α ◽  
The Impact

Abstract Reproduction of dynamic physiologic erythropoiesis in vitro requires a three-dimensional (3D) architecture, erythroblast-macrophage interactions and cytokines such as erythropoietin (EPO). The role of oxygen concentration gradients in this process is unclear. We have created a 3D bone marrow (BM) biomimicry using collagen-coated polyurethane scaffolds (5mm3) to expand cord blood mononuclear cells (CBMNCs) in a cytokine-free environment for 28 days (D). Addition of EPO to this system induces mature erythropoiesis. We hypothesised that physiologic concentrations of cytokines - stem cell factor (SCF) / EPO - and a hypoxia (H)/normoxia (N) schedule to mimic BM oxygen gradients would enhance erythropoiesis. CBMNCs were seeded (4x106 cells/scaffold) in 3D serum-free cultures supplemented with 10ng/mL SCF (D0-D28), and 100mU/mL EPO (D7-D28), with medium exchange every 3D. Three conditions were compared: N (20%), H (5%) and 2-step oxygenation HN (H D0-D7 and N thereafter). Erythroid maturation was monitored weekly by flow cytometry (CD45/CD71/CD235a) both in situ (i.e., in scaffolds) and in supernatant (S/N) cells. D0-7 H was more efficient in early induction of CD235a in the absence of exogenous EPO (H 13% vs N 8% CD45loCD71+CD235alo cells, p<0.05). This maturation profile was also observed in D10 S/N cells, in which CD45loCD71+CD235a+ cells were proportionately more in H (30%) and HN (27%) than in N (16%, p<0.05). By D14, N and HN stimulated the appearance of CD45-CD71+CD235a+ cells, whereas H maintained the CD45loCD71+CD235a-/lo phenotype. By D21, a CD45-CD71+CD235a+ mature population was clearly distinguished in all conditions, most notably in N (16%) and HN (21%) vs H (9%). At D28, more mature CD45-CD71loCD235a+ cells were observed in normoxia conditions, N 3% and HN 4%, vs H 0.3%. A renewed population of erythroid progenitors was also evident at this time (H 62%, N 51% and HN 46% CD45loCD71lo/+CD235a- cells). In order to assess the impact of H and N on erythroid gene transcription, we evaluated erythroid signatures by qRT-PCR. GATA-1 expression was detected from D7, highest for H at D14 (p<0.05), and decreased thereafter. GATA-2 expression was up-regulated only at D28, in particular in N (p<0.05), and correlated with emerging erythroid progenitors identified at this stage. At D14, EPOR expression was maximal, especially in HN (p<0.05), simultaneous with high pSTAT5 levels, suggesting activation of EPOR signalling. Also at D14, H upregulated γ-globin (p<0.05). By Western Blot, only H and HN still produced γ-globin whereas β-globin expression was clearly detected in all conditions by D28. In situ production of cytokines was evaluated by cytometric bead array in the exhausted media. IL-6, G-CSF, GM-CSF, IL-1, TNF-α and IL-17 were detected at higher concentrations during the first 7 days, declining to undetectable thereafter. IL-21 was not detected at any point. IL-3 was detected from D13, with highest expression in H (p<0.05, D22). VEGF was also expressed after D7, highest in H (p<0.05, D16 & D19), concurrent with HIF-1α up-regulation observed at D7 and D14. TNF-α was produced with variable intensity from D4. These data suggested that D7-D14 was a crucial period for culture dynamics, in particular for H and HN, with up-regulation of erythroid transcription factors, EPOR signalling, and endogenous cytokine production. BFU-E and CFU-E also dominated the first 14 days of culture. Scanning electron microscopy at D17 and D25 revealed niche-like structures in situ, which expressed STRO-1, osteopontin and vimentin at D19 by confocal immunofluorescent microscopy, indicative of an endogenous stromal cell microenvironment. CD68+ cells were also detected at D19 in proximity to CD71+ cells suggesting formation of erythroblastic islands. In this 3D ex vivo biomimicry using near-physiologic cytokine and oxygen conditions, H induced initial erythroid commitment and established an early erythroid progenitor population. N was required at later maturational stages and enhanced the γ-globin to β-globin switch. We identified D7-D14 as a crucial timeframe in this system wherein endogenous cytokine production as well as up-regulation of GATA-1, EPOR and HIF-1α was observed. We propose that a combined HN schedule in this 3D BM biomimicy may enable a more robust and physiologic culture platform to study normal and abnormal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Tailor-Made Protein Synthesis Program Drives Erythroid Development and Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3581-3581
Author(s):  
Craig M Forester ◽  
Zhen Shi ◽  
Maria Barna ◽  
Davide Ruggero
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Erythroid Cell ◽  
Translation Control ◽  
Erythroid Progenitors ◽  
Erythroid Precursor ◽  
Erythroid Development

Abstract Erythropoiesis constitutes the largest demand on the hematopoietic system due to its extraordinary production on a daily basis. The erythroid proteome requires an integration of multiple external cues to coordinate programs of differentiation as well as maintenance of erythroid precursors. The biomedical relevance of this critical process is underscored by recent findings showing impaired ribosome function in an entire class of clinical disorders with severe impairments in erythroid differentiation, known as ribosomopathies, which remain poorly understood. One of the main signaling pathways controlling post-transcriptional gene expression during erythropoiesis is the mTOR pathway. mTOR activation downstream of SCF/Epo in erythroid progenitors controls the activity of the major cap-binding protein eIF4E. However, the functional role of eIF4E during erythropoiesis and protein synthesis control in this cell type remains unexplored. Here we show that eIF4E activity, through mTOR-dependent phosphorylation of its inhibitory protein 4EBP1, unexpectedly undergoes a dynamic switch between early erythroid precursor populations and during terminal erythrocyte maturation, where eIF4E becomes progressively silenced. Employing a unique eIF4E transgenic mouse model, we strikingly show that overexpression of eIF4E in the bone marrow compartment results in an early accumulation of erythrocyte precursors and a block in erythrocyte differentiation. Surprisingly, this new role of eIF4E in erythropoiesis is independent from control of global protein synthesis but instead may promote a specialized program of translation control that is customized for erythroid cell function. Employing state of the art unbiased proteomics, our work is uncovering distinct networks of proteins, whose expression levels are controlled by eIF4E dosage during specific phases of erythrocyte maturation. Together, our research highlights a novel molecular program linking exquisite regulation of eIF4E activity to specialized translational control underlying erythroid development, providing unprecedented insight into the etiology of erythroid dysfunction in ribosomopathies. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Role of TAL1 in Hematopoiesis and Leukemogenesis

Acta Naturae ◽  
2018 ◽  
Vol 10 (1) ◽  
pp. 15-23 ◽  
Author(s):  
E. R. Vagapova ◽  
P. V. Spirin ◽  
T. D. Lebedev ◽  
V. S. Prassolov
Keyword(s):  
Transcription Factors ◽  
T Cell ◽  
Blood Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Formation ◽  
Myeloid Differentiation ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Cell Acute Lymphoblastic Leukemia

TAL1 (SCL/TAL1, T-cell acute leukemia protein 1) is a transcription factor that is involved in the process of hematopoiesis and leukemogenesis. It participates in blood cell formation, forms mesoderm in early embryogenesis, and regulates hematopoiesis in adult organisms. TAL1 is essential in maintaining the multipotency of hematopoietic stem cells (HSC) and keeping them in quiescence (stage G0). TAL1 forms complexes with various transcription factors, regulating hematopoiesis (E2A/HEB, GATA1-3, LMO1-2, Ldb1, ETO2, RUNX1, ERG, FLI1). In these complexes, TAL1 regulates normal myeloid differentiation, controls the proliferation of erythroid progenitors, and determines the choice of the direction of HSC differentiation. The transcription factors TAL1, E2A, GATA1 (or GATA2), LMO2, and Ldb1 are the major components of the SCL complex. In addition to normal hematopoiesis, this complex may also be involved in the process of blood cell malignant transformation. Upregulation of C-KIT expression is one of the main roles played by the SCL complex. Today, TAL1 and its partners are considered promising therapeutic targets in the treatment of T-cell acute lymphoblastic leukemia.

Sign in / Sign up

Export Citation Format

Share Document