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.

Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1919-1919
Author(s):  
Iman Hatem Fares ◽  
Jalila Chagraoui ◽  
Jana Krosl ◽  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. 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.

Expansion and Biased Commitment of Hematopoietic Stem Cells Towards Erythroid Cells upon Acute Anemia Induction

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1270-1270
Author(s):  
Mark van der Garde ◽  
Valgardur Sigurdsson ◽  
Visnja Radulovic ◽  
Svetlana Soboleva ◽  
Abdul Ghani Alattar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Rapid Expansion ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Transient Event ◽  
Acute Anemia

Abstract The majority of adult hematopoietic stem cells (HSCs) are maintained in a dormant state under homeostatic conditions. In contrast, under stressed conditions such as myeloablation and infection, HSCs are known to proliferate and rapidly give rise to downstream progeny. However, it is unclear whether and how HSCs respond to severe anemic conditions. Here we report that HSCs rapidly expand with a biased differentiation towards erythroid cells upon the induction of acute anemia. Injection of 60 mg/kg of phenylhydrazine (PHZ) was used to induce hemolytic anemia, after which the peripheral blood (PB), bone marrow (BM) and spleen of the mice were analyzed for the blood profiles and stem/progenitor cell content. The red blood cell (RBC) count of the PHZ treated mice was at its lowest at day 6 post injection. BM analysis showed that the number of HSCs (CD150+CD34-c-kit+Sca-I+Lineage-) immediately started increasing, as well as megakaryocyte-erythroid progenitors (MEP, CD34-FcγIII/IIR-c-kit+Sca-I-Lineage-) with a peak at day 3-4 (3.0 and 3.4 fold increase, respectively). Interestingly, the number of common myeloid progenitors (CMP, CD34+FcγIII/IIR-c-kit+Sca-I-Lineage-) did not show a clear increase over time and the number of erythroid progenitors (Ter119+) started increasing at a later time point than the HSC/MEP expansion, suggesting that the expansion of primitive cells is a primary response to the anemic condition that possibly skips some of the regular stages that are observed in the normal differentiation towards erythrocytes. In contrast to the BM, in the spleen HSC expansion was modest while MEP and CMP were robustly expanded (5.7 and 6.6 fold increase, respectively). These findings indicate that the BM and spleen have distinct roles in the response to the anemic conditions. In order to accurately evaluate the lineage potential of HSCs in vitro, we developed a combined assay utilizing colony formation and flow cytometry analysis (CFU-FACS), with which all generated colonies were analyzed for the morphology and the frequency of each lineage. The result showed that HSCs isolated from control mice had a balanced differentiation towards megakaryocyte and erythroid cells with 20-25% of the colonies containing only granulocytes/macrophages and megakaryocytes, but not erythroid cells (GMMk colonies). In contrast, HSCs isolated from PHZ treated mice showed significantly increased the number of colonies containing a higher content of erythroid cells, whereas the ratio of GMMk colonies was decreased. Furthermore, 3-dimensional analysis of the three lineage potentials (myeloid, megakaryocyte and erythroid) in the colonies revealed an imbalanced lineage potential of HSCs from anemic mice, showing higher erythroid potential instead of the megakaryocyte potential. As an alternative method, phlebotomy was performed to induce acute anemia. Although phlebotomized mice did not display a clear expansion of the HSC population, CFU-FACS analysis showed an erythroid-biased lineage potential of the HSCs, indicating that the HSC expansion and the lineage bias may be caused by independent mechanisms. To demonstrate if the alterations in the HSCs affect the in vivo function of these cells, 50 HSCs isolated from control or PHZ injected Kusabira Orange (KuO) mice were transplanted into lethally irradiated mice. Two weeks after the transplantation, the ratio of KuO+ RBCs against KuO+ platelets was higher in the PHZ-HSC transplanted mice than control-HSC transplanted mice. This difference was not seen four weeks after transplantation and the long-term reconstitution (>12 weeks) levels did not differ between both groups, suggesting that the enhanced erythropoiesis is a transient event that does not reduce the stem cell capacity. In summary, we demonstrated that not only progenitor cells but also HSCs respond to severe anemic conditions and contribute to erythropoiesis through rapid expansion and a transient fate change, depicting a novel model of stress response. Disclosures No relevant conflicts of interest to declare.

The Final Cellular Output in Human Erythroid Massive Amplification Culture (HEMA) Is Determined by Dynamic Interactions Between Immature and Mature Cell Populations.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3156-3156 ◽  
Author(s):  
Massimo Sanchez ◽  
Francesca Masiello ◽  
Valentina Tirelli ◽  
Barbara Ghinassi ◽  
Carolyn Whitsett ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Cell Number ◽  
Proliferation Index ◽  
Dynamic Interactions ◽  
Relative Contribution ◽  
Myeloid Progenitor Cells

Abstract Abstract 3156 Poster Board III-93 Hematopoietic progenitor cells cultured with stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO) generate erythroid cells that mature in 2 weeks. Addition of dexamethasone and estradiol retards maturation providing the basis for the development of HEMA, a system that generates ex vivo human erythroblasts (EBs) in numbers sufficient for transfusion. In HEMA, EBs at different stages of maturation coexist for > 21 days. FACS analyses for thrombospondin receptor (CD36) and Glycophorin A (CD235a) divides EBs into 4 maturation classes: CD36highCD235aneg (CFU-E) and CD36highCD235alow (pro-EBs) (immature EBs, iEBs); CD36highCD235ahigh (basophilic-polychromatic EBs) (mature EBs) and CD36lowCD235ahigh (orthochromatic EBs) cells. At day 10, 5-19% of the cells are non-erythroid CD36negCD235aneg cells while orthochromatic EBs are barely detectable at any time. The aim of this study was to identify the relative contribution of different populations to the final EBs output of HEMA. The cells present at day 10 in HEMA established with adult mononuclear cells were separated by sorting, labeled with CSFE and cultured for 4 additional days for fate determination (proliferation, maturation and/or death). To identify which population had the ability to generate more EBs, unfractionated cells and cells isolated by sorting (non-erythroid, iEBs and mEBs) were cultured either in colony assay or HEMA. Ninety-three percent of the colony forming ability (all CFU-GM) of unfractionated cells was recovered in the CD36negCD235aneg fraction which did not grow in HEMA. However, iEBs did not contain colony forming cells but generated twice as many EBs as unfractionated cells [3.9±1.0 vs 2.38±0.32 fold increase (FI), respectively). In cultures maintained for 16 days, iEBs sequentially sorted every 2 days generated 5-fold more EBs than the corresponding unfractionated EBs (FI=25 vs 5, respectively). mEBs did not generate colonies or proliferate but died in HEMA. Calculations of hemi CSFE staining decrements indicated that the division index of iEBs and mEBs in the first 24 hrs of HEMA was 1.32±0.08 and 0.4±0.08 (p<0.01), respectively. The higher number of cells generated by iEBs with respect to unfractionated cells suggested that mEBs may inhibit iEBs proliferation. To characterize this effect, iEBs and mEBs were co-cultured for 48 hrs at ratios of 100/0, 5/1, 5/3 and 1/2. One of the two populations was labelled by CSFE staining. To take into account a possible toxic effect of CSFE-labeling, experiments were repeated with either CSFE-labelled iEBs or CSFE-labelled mEBs measuring proliferation/maturation/apoptosis of labeled and unlabelled cells, respectively. iEBs alone increased in numbers by 2-fold over 4 days. By contrast, iEB-derived cells did not increase in number in co-cultures at 5/1 and 5/3 mEB ratios and were undetectable in those at 1/2 mEB ratio. Although cell number did not increase in the co-cultures, the proliferation index (1.3), levels of maturation (50% newly generated mEBs) and apoptosis (barely detectable Annexin Vpos cells) of iEBs in co-cultures remained similar to those observed when these cells were cultured alone. The failure to detect cells after 4 days in iEB/mEB co-cultures at 1/2 ratio suggests that mEBs induce non-apoptotic iEB death. In conclusion, three cells populations were present at day 10 in HEMA of adult mononuclear cells: a CD36negCD235aneg population that contains myeloid progenitor cells and does not generate EBs; iEBs, that generate additional iEBs as well as mEBs; and mEBs that have low proliferation activity, limited life-span and induce non-apototic iEB death. Therefore, the final cellular output of HEMA is determined by a delicate balance between proliferation, maturation and cell death of iEBs and mEBs. Disclosures No relevant conflicts of interest to declare.

Effects of Ontogeny, Ethnicity, Gender and Loss of Companion Cells on Ex-Vivo Expansion of Erythroid Cells for Transfusion

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1261-1261
Author(s):  
Valentina Tirelli ◽  
Francesca Masiello ◽  
Massimo Sanchez ◽  
Barbara Ghinassi ◽  
Carolyn Whitsett ◽  
...  
Keyword(s):  
African American ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Individual Variability ◽  
Erythroid Cells ◽  
Companion Cells ◽  
Mother Daughter Relationship ◽  
Cell Sources ◽  
Donor Variability

Abstract Abstract 1261 The observation that CD34pos and mononuclear cells (MNC) from cord (CB) and adult (AB) blood generate great numbers of erythroid cells (EBs) in human erythroid massive amplification (HEMA) cultures (cultures stimulated with human SCF, IL-3, erythropoietin, dexamethasone and estradiol) led to the hypothesis that EBs generated ex-vivo from currently discarded stem cell sources (low volume CB and MNC from leukoreduced AB) may serve as transfusion products. Studies on ex-vivo EB expansion performed primarily with cells from de-identified Caucasian donors have demonstrated donor variability on the number of EBs generated. Alloimmunized patients of African descendent often require antigen-matched transfusion products which can only be found among ethnically-matched donors. The aim of this study was to determine how the variability in ex-vivo EB expansion under HEMA conditions is affected by ontogeny, ethnicity, gender and loss of companion cells during preparation procedures. Effect of ontogeny and ethnicity: EBs expansions in 3 replicate cultures from the same AB (3 donors) differed only by 10%. By contrast, average day 14 FI in HEMAser cultures from 27 AB (26 Caucasians and 1 African-american) and 7 CB (all Caucasians) varied widely. By day 15, FI ranged from 1.7–30 for AB (FI=30 for the African-american AB) and 7.5–337 for CB. The average FI observed with CB was greater than that observed with AB (51±63 vs 12±10, p<0.05) in spite of similar colony forming cell (CFC) content [123±82 (n=5) vs 73±28 (n=18) CFC/105 MNC). Effect of gender: AB MNC from females (n=3) contained lower numbers of CFC than those from males (n=3)(79±35 vs 164±36 total CFC/105 MNC, p<0.05). However, day 15 FI observed in the corresponding HEMAser (11±3 vs 11±7) and HEMAdef (media formulated with human components, 80±20 vs 82±10) cultures were similar. The FI observed with AB under HEMAdef conditions are not statistically different from those obtained from CB. Effect of loss of companion cells: Both AB and CB MNC generate ∼10-times more EBs than CD34pos cells. This effect may be ascribed either to loss of CD34neg erythroid precursors (van den Akker et al. Haematologica, 95:1594, 2010) and/or to the fact that current purification procedures recover only 30–50% of the total number of CD34pos cells present in MNC. To clarify the effect of loss of companion cells (CD34pos and/or CD34neg) on EB expansion, the frequency and EB expansion potential of cells sorted according to the CD34/CD36 profile from AB and CB were compared. Total MNC were cultured in parallel as control. In AB, 3 populations (A, CD34posCD36neg, 0.1±0.05%; B, CD34posCD36pos, barely detectable-0.1% and C, CD34negCD36low, 2±0.8%) with EBs expansion potential were identified. Populations A, B and C generated EBs with day 15 FI of ∼9,500, 113 and 60, respectively which matured (>10% CD235ahigh) by day 8, 7 and <7. The frequency of B and C cells transiently increased (up to 0.3–0.9%) during the first 6 days of HEMAser culture of population A. These results suggest that population A, B and C are linked in a mother-daughter relationship and that transition from A to C involves major losses in expansion potential. Due to this loss of expansion potential, the contribution of CD34neg cells to the total number of EBs generated from AB MNC is negligible. Calculations of the total EB numbers generated from an entire AB unit and from individual purified fractions indicated that AB MNC generated more EBs than the purified fractions in combination (total EBs numbers=2.7×109 vs 7×108). Therefore, for AB, loss of CD34pos cells, rather than of CD34neg precursors, is the main reason for the lower numbers of EBs generated by CD34pos cells than MNC. Population A (1.3±0.9%), B (0.4±0.3%) and C (6.8±4.8%) were also recognized in CB but they generated similar numbers of EBs (day 16 FI ∼600 in all cases). Since in CB, transition from population A-B and C was not associated with restriction in proliferation potential, the greater numbers of EBs generated by MNC than by CD34pos cells are due both to loss of CD34neg (CD36low) precursors and of CD34pos cells during purification. These studies indicate that, in spite of individual variability in EBs expansion, cell losses during sample processing and media used for expansion have greater impact on the number of EBs expanded ex-vivo than demographic properties (ontogeny, ethnicity and/or gender) of the donor. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Humanized Culture Medium for Clinical Expansion of Human Erythroblasts

2010 ◽  
Vol 19 (4) ◽  
pp. 453-469 ◽  
Author(s):  
Giovanni Migliaccio ◽  
Massimo Sanchez ◽  
Francesca Masiello ◽  
Valentina Tirelli ◽  
Lilian Varricchio ◽  
...  
Keyword(s):  
Growth Factors ◽  
Cord Blood ◽  
Stem Cell Factor ◽  
Culture Medium ◽  
Mononuclear Cells ◽  
Culture Media ◽  
Ex Vivo ◽  
Fold Increase ◽  
Human Albumin ◽  
Clinical Use

Ex vivo-generated erythroblasts represent alternative transfusion products. However, inclusion of bovine components in media used for their growth precludes clinical use, highlighting the importance of developing culture media based on pharmaceutical grade reagents. In addition, because adult blood generates ex vivo lower numbers of erythroblasts than cord blood, cord blood has been proposed as the source of choice for ex vivo erythroblast production. To clarify the potential of adult blood to generate erythroblasts ex vivo, experiments were designed to identify growth factors [stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and/or thrombopoietin (TPO)] and the optimal concentration and addition schedule of hormones (dexamethasone and estradiol) sustaining maximal erythroid amplification from adult blood mononuclear cells (MNC) 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 a 6–12-fold increase in erythroid cells while TPO was ineffective. Dexamethasone and estradiol (both at 10−6 M) exerted partially overlapping but nonredundant functions. Dexamethasone was indispensable in the first 10 days of culture while estradiol was required from day 10 on. The growth factor and hormone combinations identified in HEMAser were then used to formulate a media composed of dialyzed pharmaceutical grade human albumin, human albumin-lipid liposomes, and iron-saturated recombinant human tranferrin (HEMAdef). HEMAdef sustained erythroid amplification as efficiently as HEMAser for cord blood MNC and 10-fold higher than HEMAser for adult blood MNC. In fact, the numbers of erythroblasts generated in HEMAdef by adult MNC were similar to those generated by cord blood MNC. In conclusion, this study identifies growth factors, hormone combinations, and human protein-based media that allow similar levels of ex vivo erythroid expansion from adult and cord blood MNC, paving the way to evaluate adult blood as a source of ex vivo-expanded erythroblasts for transfusion.

scholarly journals Interferon Induction By HbS, SERINC5 Upregulation and Reduction of HIV-1 Nef and AP2 Contribute to HIV-1 Restriction in Sickle Cell Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-6
Author(s):  
Namita Kumari ◽  
Marina Jerebtsova ◽  
Songping Wang ◽  
Sharmin Diaz ◽  
Sergei Nekhai
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Interferon Response ◽  
Cell Disease ◽  
Restriction Factors ◽  
Peripheral Blood Mononuclear ◽  
Hiv 1

Concerted action of numerous positively acting cellular factors is essential for Human immunodeficiency virus type 1 (HIV-1) replication but in turn is challenged by anti-viral restriction factors. Previously we showed that ex vivo one round HIV-1 replication and replication of fully competent T-tropic HIV-1(IIIB) is significantly reduced in peripheral blood mononuclear cells (PBMCs) obtained from patients with Sickle Cell Disease (SCD). Further, we identified and confirmed CDKN1A (p21) and CH25H as host restriction factors expressed in SCD PBMCs that may contribute to the HIV-1 inhibition, in addition to the previously reported SAMHD1 and IKBα. Since CH25H is an interferon stimulated gene (ISG), we analyzed IRFs and interferon expression in SCD PBMCs. Higher levels of IRF7 and IFNβ mRNA were observed in SCD PBMCs compared to controls. We probed further to ascertain if hemin or sickle Hb was responsible for interferon response. We found upregulation of IFNβ in THP-1 - derived macrophages treated with lysates of HbSS RBCs or purified HbS as compared to untreated or HbA treated controls. HbSS RBCs lysates and purified HbS inhibited HIV-1 gag mRNA expression in monocyte-derived macrophages infected with HIV-1(Ba-L). Recent clinical study showed increased levels of CD4 in HIV-1 infected SCD patients in Africa. Thus we analyzed CD4 levels in HIV-1 IIIB infected SCD PBMCs, and found them to be higher compared to controls. Levels of HIV-1 nef mRNA, that controls CD4 expression was lower in HIV-1 IIIB infected SCD PBMCs. As Nef counteracts SERINC3/5 restriction factor, we analyzed its expression as well as the expression of AP2 clathrin adaptor that is required for Nef mediated internalization of CD4. AP2 expression was lower and SERINC5 expression was higher in SCD PBMCs. CONCLUSIONS: SCD PBMCs could resist HIV-1 infection because of the increased IFNβ production by macrophages exposed to HbSS or sickle cell RBCs. SCD PBMC have increased levels of SERNIC5 and lower levels of HIV-1 Nef and host AP2 expression that, culumlatively, can increased CD4 levels and lead to the overall improved immunological health of SCD patients. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 1SC1HL150685, 5U54MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures No relevant conflicts of interest to declare.

Efficient Erythropoiesis From Human Embryonic Stem Cells Through Dimerization of Intracellular MPL.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2291-2291
Author(s):  
William Sang Kim ◽  
Gautam G. Dravid ◽  
Yuhua Zhu ◽  
Chintan Parekh ◽  
Qiming Deng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Total Yield ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Akt Signaling ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Enucleated Rbc ◽  
Negative Controls

Abstract Abstract 2291 Objectives: Unlimited self renewal capacity and the ability to differentiate into any cell type make human pluripotent stem cells (PSC) a potential source for the ex vivo manufacture of red blood cells (RBC) for safe transfusion. Current methods of RBC differentiation from PSC suffer from low yields of RBCs, most of which contain embryonic rather than adult or fetal hemoglobins. Therefore, efficient clinical translation of this strategy is critically dependent on the development of novel methods to enhance the generation of functional RBCs from PSC. We have previously shown that dimerization of the intracellular component of MPL (the thrombopoietin receptor), induces expansion of myelo-erythroid progenitors (MEP) from human cord blood as well as their terminal differentiation into enucleated RBC through unique, EPO-independent mechanisms (Parekh et al, 2012). Our goal was to investigate the potential of intracellular MPL dimerization to induce erythropoiesis from human PSC and to identify the signaling pathways activated by this strategy. Methods: Human embryonic stem cell (hESC) lines H1 and HES3 were transduced with a lentiviral vector to express the fusion protein F36V-MPL (containing the ligand binding domain F36V and the intracytoplasmic portion of MPL). Dimerization of F36V-MPL was accomplished by addition of the synthetic ligand AP20187 (aka CID) during culture (with or without erythropoietin) on OP9 stroma in the absence of other cytokines. F36V-MPL transduced-hESC that did not receive CID and F36V-transduced hESC cultured with CID served as negative controls. Flow cytometry and Colony Forming Unit (CFU) assays were used to analyze erythroid differentiation. Phosflow and Western Blot were used to analyze cell signaling. MEP generated during hESC differentiation were defined as cells co-expressing GlyA and CD41a/CD42a. Results: F36V-MPL dimerization induced significantly more Glycophorin A+ cells (P=0.0001; n=5) and 10-fold higher number of erythroid CFU (P=0.0007; n=15) as compared to negative controls. The effect was consistent across different hESC cell lines. The increased yield of erythroid cells was not due to an overall increase in cell proliferation as the total yield of cells was not statistically different between treated and untreated cultures. This effect was seen in the absence of any hematopoietic cytokines, including erythropoietin (EPO), a critical cytokine for erythropoiesis and an integral component of all ex vivo PSC erythroid differentiation protocols, indicating that MPL dimerization alone is sufficient to induce erythropoiesis from hESCs. Erythroid output was further enhanced in an additive manner in the presence of EPO (P=0.0058; n=5). In order to identify the point at which MPL dimerization affects erythropoiesis, CID was added during differentiation directly from hESC or to isolated MEP generated from hESC. CID and EPO increased the number of MEP compared to untreated controls, demonstrating that MPL dimerization induces the generation of early erythroid progenitors. In addition, CID drove erythroid differentiation from MEP more efficiently than EPO, demonstrated by a significantly higher frequency of total erythroid cells (P=0.02; n=3), and 4-fold increase in yield of enucleated RBC. This indicates that CID has a greater effect on terminal erythroid differentiation than EPO. We then investigated the signaling mechanism activated by F36V-MPL dimerization and found that, unlike the full-length MPL receptor, which activates both STAT5/JAK2 and AKT pathways, F36V-MPL dimerization activated AKT but not STAT5 or JAK2 phosphorylation. PI3K/AKT inhibitors (LY294002 and AKT inhibitor IV) effectively inhibited erythroid differentiation of transduced hESC cultured in the presence of CID (P=0.0442; n=2) indicating that MPL dimerization induced erythropoiesis is dependent on AKT signaling. Conclusion: F36V-MPL dimerization during hESC-derived hematopoiesis induces EPO-independent erythroid differentiation through AKT signaling, by both generating erythroid progenitors and promoting maturation of RBC. MPL dimerization also is more potent than EPO in inducing erythropoiesis from hESC and has an additive effect when combined with EPO, making this a potential strategy for the generation of therapeutically relevant levels of functional enucleated RBCs from PSC. Disclosures: No relevant conflicts of interest to declare.

Robust Induction Of Th1-Like γδ T Cells With Anti-Myeloma Activity By Lenalidomide In Combination With HMB-PP As Well As Zoledronic Acid

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 129-129
Author(s):  
Takeshi Harada ◽  
Qu Cui ◽  
Shingen Nakamura ◽  
Hirokazu Miki ◽  
Asuka Oda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Zoledronic Acid ◽  
Cytotoxic Activity ◽  
Immune Cells ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Γδ T Cells ◽  
Bone Marrow Microenvironment

Abstract Multiple myeloma (MM) still remains incurable even with the implementation of novel therapeutic modalities, leading to the idea to develop various forms of immunotherapies. In this regard, γδ T cells bearing Vγ9Vδ2 TCR expanded from peripheral blood mononuclear cells (PBMCs) have attracted attention as potent effectors available in a novel immunotherapy against MM. Human Vγ9Vδ2 γδ T cells can be expanded ex vivo by aminobisphosphonates in combination with IL-2, and effectively target and impair MM cells. However, MM cells appear to protect themselves from external insults by immune cells in a unique bone marrow microenvironment created by the accumulation of mesenchymal stem cells/bone marrow stromal cells (BMSCs) with defective osteoblastic differentiation and acid-producing osteoclasts. To improve the therapeutic efficacy of γδ T cells, therefore, we need to develop a maneuver to effectively enhance the expansion and activity of γδ T cells while disrupting the MM cell-bone marrow interaction. Lenalidomide (Len), a novel immunomodulatory anti-MM agent, shows pivotal anti-MM activity by targeting immune cells as well as the interaction of MM cells and their surrounding cells in the bone marrow. The present study was undertaken to explore the efficacy of Len in combination with zoledronic acid (Zol) or a precursor of isopentenyl pyrophosphate (IPP) (E)-4 hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a microbial antigen for Vγ9Vδ2 TCR, on the induction and expansion of Th1-like γδ T cells with enhanced cytotoxic activity against MM cells in the skewed bone marrow microenvironment in MM. When combined with Zol (1μM), clinically relevant doses of Len (around 1 μM) substantially expanded γδ T cells from PBMCs to the levels similar to IL-2 (100 U/ml). Len was able to expand γδ T cells more robustly in combination with HMB-PP (1 μM) than Zol from PBMCs from the majority of normal donors. However, Len alone did not show any significant effects on γδ T cell expansion and activation, suggesting a costimulatory role of Len on Zol or HMB-PP-primed γδ T cells. The surface expression of LFA-1, and the cytotoxicity-associated molecules NKG2D, DNAX accessory molecule-1 (DNAM-1; CD226) and TRAIL were up-regulated in the expanded γδ T cells. Although functional diversity has been demonstrated in γδ T cells expanded by various stimuli, Len in combination with either Zol or HMB-PP enhanced intracellular IFN-γ along with the surface NKG2D but not Foxp3 in γδ T cells at higher levels than IL-2, suggesting robust induction of Th1-like γδ T cells by Len. Importantly, γδ T cells expanded with the combinatory treatments with Len and Zol or HMB-PP exerted potent cytotoxic activity against MM cells but not normal cells surrounding MM cells in bone marrow samples from patients with MM. Such treatments with Len was able to maintain the cytotoxic activity of the γδ T cells against MM cells in acidic conditions with lactic acid, and restored their anti-MM activity blunted in the presence of BMSCs. Interestingly, the expanded γδ T cells markedly suppressed the colony formation in semi-solid methylcellulose assays of RPMI8226 and KMS-11 cells [81±1 (mean ± SD) vs. 0±0 and 40±1 vs. 16±4 colonies/dish, respectively, p<0.01], and decreased in size their side populations, suggesting targeting a drug-resistant clonogenic MM cells. These results collectively demonstrate that Len and HMB-PP as well as Zol are an effective combination for ex vivo expansion of Th1-like γδ T cells with potent anti-MM activity, and suggest that Len in combination with Zol may maintain their in vivo anti-MM activity in the bone marrow where MM cells reside. The present results warrant further study on Len-based immunotherapy with γδ T cells. 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.

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