Leukocyte Immunoglobulin-Like Receptor B Regulates Erythroblastic Island Size in Human in-Vitro Erythroblastic Islands

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3190-3190
Author(s):  
Ajay Perumbeti ◽  
Alin Girnita ◽  
Jose A. Cancelas
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Close Contact ◽  
Surface Receptors ◽  
Erythroblastic Island ◽  
Erythroid Precursors ◽  
Surface Receptor

Abstract Abstract 3190 Erythropoiesis occurs in a unique bone marrow (BM) microenvironment known as erythroblastic islands. Erythroblastic islands are composed of erythroid precursors in close contact with specialized stromal macrophages, also referred to as central or island macrophages. Human erythroblastic island macrophages have been difficult to discern from other macrophage subpopulations by cell surface markers, although they have reduced expression of C3b receptor (CD35) (Lee et al., 1988). The unique non-antigen presenting nature of erythroblastic island macrophages led us to hypothesize that it may share surface receptors of other tolerogenic macrophages subpopulations, including leukocyte immunoglobulin-like transcript B receptors (LILRB). We first identified a distinct subset of human bone marrow macrophages with flow cytometry, which express high amounts of LILRB1 and LILRB2 surface receptors, have low expression of C3b surface receptor (p<0.05), and have increased expression of VCAM-1 surface receptor. This macrophage subpopulation consisted of 1 in 500 (0.02%) of bone marrow mononuclear cells. Functionally, when we FACS sorted human BM enriched erythroid precursors (CD45−/CD71+) and combined them with FACS sorted LILRB1+/LILRB2+ (LILRB+) or LILRB1−/LILRB2- (LILRB−) BM macrophages, there were no significant differences in erythroblastic island reconstitution, suggesting that the adhesion of primary BM erythroblasts to island macrophages is not related to macrophage LILRB expression. Since our ex-vivo erythroid population was enriched for late precursors, we tested island reconstitution with CD34+ cell-derived cultured erythroblasts and FACS sorted LILRB+ or LILRB- BM macrophages, which resulted in a significantly higher number of large proliferative erythroblastic islands (>25 erythroblasts/island macrophage) in LILRB+ compared with LILRB- macrophages (p<0.05) (Figure 1). Incubation with neutralizing anti-LILRB1/LILRB2 antibodies did not prevent primary erythroblastic island reconstitution independent of expression of LILRB by macrophages. However, anti-LILRB1/LILRB2 did prevent the formation of large erythroblastic islands indicating a specific role of LILRB in optimal island reconstitution and erythropoiesis (p<0.05) (Figure 1). In conclusion, we demonstrate that LILRB+ BM macrophages increase the formation of large in-vitro erythroblastic islands and LILRB receptors are important candidate receptors for functional regulation of erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

Role of PDGF/PDGFR in Regulation of Thrombopoiesis: A Possible Explanation for Imatinib Mesylate-Induced Thrombocytopenia in the Treatment of CML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3348-3348
Author(s):  
Mo Yang ◽  
Fanyi Meng ◽  
Jie yu Ye ◽  
Yue Xu ◽  
Bin Xiao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Imatinib Mesylate ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Potential Effect ◽  
Mice Model ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Bone Marrow Histology

Abstract Abstract 3348 Platelet-derived growth factor (PDGF), a platelet alpha-granule molecule, imply their potential effect in the regulation of megakaryocytopoiesis and thrombopoiesis, which also intimates the existence of an autocrine and/or paracrine loop constructed by megakaryocytes/platelets and their granular constituents. Our previous studies demonstrated the presence of functional PDGF receptors (PDGFR) on human megakaryocytes and platelets (Yang et al, Thromb Haemastasis, 1997) and CD34+ cells, and their ability to mediate a mitogenic response. PDGF promoted the ex vivo expansion of human hematopoietic stem (CD34+) and progenitor (CD41+ CD61+) cells. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in NOD/SCID mice. PDGF stimulated in vitro megakaryocytopoiesis via PDGFR and/or the indirect effect on bone marrow microenvironment to produce TPO and other cytokines. It also showed a direct stimulatory effect of PDGF on c-Fos, GATA-1 and NF-E2 expressions in megakaryocytes. We speculate that these transcription factors might be involved in the signal transduction of PDGF on the regulation of megakaryocytopoiesis. PDGF also enhanced platelet recovery in mice model with radiation-induced thrombocytopenia. Studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow colony-forming unit-megakaryocyte (CFU-MK) in this irradiated-mouse. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow histology sections. In the M-07e apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on megakaryocytes. We also demonstrated that PDGF activated the PI3k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings suggested that the PDGF-initiated radioprotective effect is likely to be mediated via PDGF receptors with subsequent activation of the PI3k/Akt pathway. The study provides a possible explanation that blockage of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia in the treatment of CML. 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.

scholarly journals Impact of Erythropoietin Production by Erythroblastic Island Macrophages on Homeostatic Murine Erythropoiesis

2020 ◽  
Vol 21 (23) ◽  
pp. 8930
Author(s):  
Genève Perron-Deshaies ◽  
Philippe St-Louis ◽  
Hugo Romero ◽  
Tatiana Scorza
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Confocal Microscopy ◽  
Erythroblastic Island ◽  
Pathological Conditions ◽  
Erythropoietin Production ◽  
Erythroid Precursors ◽  
Clodronate Liposomes

Erythropoietin (EPO) is an essential hormone for erythropoiesis, protecting differentiating erythroblasts against apoptosis. EPO has been largely studied in stress or pathological conditions but its regulatory role in steady state erythropoiesis has been less documented. Herein, we report production of EPO by bone marrow-derived macrophages (BMDM) in vitro, and its further enhancement in BMDM conditioned with media from apoptotic cells. Confocal microscopy confirmed EPO production in erythroblastic island (EBI)-associated macrophages, and analysis of mice depleted of EBI macrophages by clodronate liposomes revealed drops in EPO levels in bone marrow (BM) cell lysates, and decreased percentages of EPO-responsive erythroblasts in the BM. We hypothesize that EBI macrophages are an in-situ source of EPO and sustain basal erythropoiesis in part through its secretion. To study this hypothesis, mice were injected with clodronate liposomes and were supplied with exogenous EPO (1–10 IU/mouse) to evaluate potential rescue of the deficiency in erythroid cells. Our results show that at doses of 5 and 10 IU, EPO significantly rescues BM steady state erythropoiesis in mice deficient of macrophages. We propose existence of a mechanism by which EBI macrophages secrete EPO in response to apoptotic erythroblasts, which is in turn controlled by the numbers of erythroid precursors generated.

JAK2 Inhibition with TG101348 Inhibits Monosomy 7 Myelodysplastic Syndromes (MDS) Bone Marrow Cells In Vitro: a Potential Targeted Therapy for Monosomy 7 MDS

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 973-973 ◽  
Author(s):  
Matthew J. Olnes ◽  
Andrea Poon ◽  
Zachary Tucker ◽  
Neal S. Young ◽  
Elaine M Sloand
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Dependent Manner ◽  
Monosomy 7

Abstract Abstract 973 The myelodysplastic syndromes (MDS) are bone marrow disorders characterized by cytopenias and a variable risk of progression to acute myeloid leukemia (AML). Monosomy 7 is the second most common cytogenetic abnormality in MDS, and the most frequent karyotypic aberration occurring in aplastic anemia patients following immunosuppressive therapy. Monosomy 7 MDS carries a particularly poor prognosis, with patients manifesting severe cytopenias and a high propensity to develop treatment-refractory AML. There are currently no targeted therapies for this disorder. We previously reported that monosomy 7 bone marrow mononuclear cells (BMMNCs) express high levels of a differentiation-defective granulocyte colony stimulating factor (G-CSF) receptor isoform (IV), an alternative splice variant that exhibits constitutive signaling through the JAK-2 and STAT-1 pathway, while levels of STAT-3 and -5 are unchanged (Sloand et al, PNAS, 2006, 103:14483). As a result, the cell's ability to differentiate is limited, while its ability to proliferate remains intact. Here we examine the effects of the highly selective JAK2 inhibitor TG101348 on monosomy 7 aneuploidy in BMMNCs, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of BMMNCs with TG101348 for 5 days significantly decreased absolute numbers of monosomy 7 aneuploid cells in a concentration dependent manner versus vehicle- treated controls (0.187 × 106 vs 1.08 × 106, P=0.007), while diploid cell numbers remained stable (0.338 × 106 vs 0.213 × 106, P=0.50). Flow cytometry experiments demonstrated that incubation with increasing concentrations of TG101348 decreased the absolute number of CD34+CD13- stem cells, and increased numbers of more differentiated CD34-CD13+ myeloid cells, with median CD34+/CD13+ ratios of 6.547 and 2.216 for cells treated with vehicle and 100 nM TG101348, respectively. By immunoblot, STAT-1 protein expression in monosomy 7 BMMNCs treated with 1uM TG101348 was decreased relative to vehicle- treated controls, while there was no difference in STAT-3 and STAT-5 levels. Thus TG101348 decreases monosomy 7 MDS blasts in vitro through inhibition of JAK-2/STAT-1 signaling, a finding that warrants further study of this agent in clinical trials for patients with monosomy 7 MDS and AML. 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.

Primary Bone Marrow-Derived MSC Subsets Have Distinct Wnt-Signatures Compared to Conventionally Cultured MSC and Differ in Their Capacity to Support Hematopoiesis in Vitro,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3414-3414 ◽  
Author(s):  
Marijke W Maijenburg ◽  
Marion Kleijer ◽  
Kim Vermeul ◽  
Erik P.J. Mul ◽  
Floris P.J. van Alphen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Wnt Signaling ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Wnt Proteins

Abstract Abstract 3414 Mesenchymal stromal cells (MSC) are of promising therapeutic use to suppress immunogenic responses following transplantation, and to support expansion of hematopoietic stem- and progenitors cells (HSPC) from small transplants derived for instance from cord blood. Culture-expanded MSC produce a wide variety and quantity of Wnt-proteins and the crucial role of Wnt-signaling in the hematopoietic stem cell niche is well established. However, studies addressing Wnt-signaling in MSC have (i) only focused on culture-expanded MSC and (ii) did not discriminate between phenotypically distinct subpopulations which are present in bulk cultures of expanded MSC. Recently we identified three new subpopulations of MSC in human bone marrow (BM) based on expression of CD271 and CD146: CD271brightCD146−, CD271brightCD146+, CD271−CD146+. These fractions co-express the “classical” MSC markers CD90 and CD105 and lack expression of CD45 and CD34 (Maijenburg et al, Blood 2010, 116, 2590). We and others demonstrated that the adult BM-derived CD271brightCD146− and CD271brightCD146+ cells contain all colony forming units-fibroblasts (Maijenburg et al, Blood 2010, 116, 2590; Tormin et al, Blood 2010, 116, 2594). To investigate how these primary subsets functionally compare to conventional, culture-expanded MSC, we investigated their Wnt-signature and hematopoietic support capacity. To this end, we sorted CD271brightCD146− and CD271brightCD146+ cells from human adult BM (n=3) and compared their Wnt-signatures obtained by Wnt-PCR array to the profiles from cultured MSC from the same donors. Fifteen genes were consistently differentially expressed in the two sorted uncultured subsets compared to their conventionally cultured counterparts. Expression of CCND1, WISP1 and WNT5B was strongly increased, and WNT5A was only detected in the conventionally cultured MSC. In contrast, WNT3A was exclusively expressed by sorted primary CD271brightCD146− and CD271brightCD146+ cells, that also expressed higher levels of JUN, LEF1 and WIF1. The differences in Wnt (target)-gene expression between CD271brightCD146− and CD271brightCD146+ cells were more subtle. The Wnt-receptors LRP6 and FZD7 were significantly higher expressed in CD271brightCD146+ cells, and a trend towards increased expression in the same subset was observed for CTNNB1, WNT11 and MYC. When the sorted subsets were cultured for 14 days (one passage), the differences in Wnt-related gene expression between the subsets was lost and the expanded sorted cells acquired an almost similar Wnt-signature as the MSC cultured from BM mononuclear cells from the same donors. The cultured subsets lost the expression of Wnt3a and gained the expression of Wnt5a, similar to the unsorted MSC cultured from the same donors in parallel. Despite the loss of a distinct Wnt-signature, co-culture experiments combining the sorted MSC subsets with human HSPC revealed that CD271brightCD146+ cells have a significantly increased capacity to support HSPC in short-term co-cultures (2 weeks) compared to CD271brightCD146− cells (p<0.021, n=3), which was analyzed in hematopoietic colony assays following co-culture. In contrast, a trend towards better long-term hematopoietic support (co-culture for 6 weeks) was observed on CD271brightCD146− cells. In conclusion, we demonstrate for the first time that primary sorted uncultured MSC subsets have a distinct Wnt-signature compared to cultured unsorted MSC and display differences in hematopoietic support. As it was recently shown that CD271brightCD146− and CD271brightCD146+ MSC localize to separate niches in vivo (Tormin et al, Blood 2011), our data indicate that the two MSC subsets are not necessarily distinct cell types and that the different Wnt-signature may be a reflection of these distinct microenvironments. Cell culturing for only one passage dramatically changed the Wnt-signature of the sorted MSC subsets, indicating that Wnt-signaling in in vitro expanded MSC does not resemble the Wnt-signature in their tissue resident counterparts in vivo. Disclosures: No relevant conflicts of interest to declare.

Role of Pleckstrin 2 in Improved Erythropoiesis of Transferrin-Treated Beta-Thalassemic Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 755-755 ◽  
Author(s):  
Maria Feola ◽  
Andrea Zamperone ◽  
Weili Bao ◽  
Tenzin Choesang ◽  
Huihui Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Actin Binding ◽  
Hematopoietic Stem ◽  
Cytoskeleton Reorganization ◽  
Erythroid Precursors

Abstract Erythropoiesis is a process during which multipotent hematopoietic stem cells proliferate, differentiate and ultimately produce enucleated reticulocytes. Terminal erythroid differentiation begins at the morphologically recognizable pro-erythroblast (pro-E) stage and is completed when orthochromatic erythroblasts (ortho-E) expel their nuclei to produce reticulocytes. Progressive differentiation between these stages occurs in homologous cell division progressively doubling proportions of pro-E, basophilic (baso-E), polychromatophilic (poly-E), and ortho-E, and multiple signaling pathways are involved in the generation of enucleated erythroid cells, including multiple steps requiring actin cytoskeleton reorganization. We have previously shown that β-thalassemic mice (th1/th1) demonstrate a disordered progression from pro-E to baso-E and that exogenous transferrin therapy restores normal proportion of early stage erythroid precursors in th1/th1 mice (Liu Blood 2013). To identify genes that play novel function in different stages of terminal erythropoiesis, we performed RNA seq analysis of sorted bone marrow pro-E from WT, th1/th1, and transferrin-treated th1/th1 mice. We identify pleckstrin-2 (plek2) as a gene of interest with a 15-fold increase in plek2 mRNA expression in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. Plek2 is an actin binding protein, like pleckstrin-1, contains a central DEP domain known to bind RacGTPase, is Epo dependent, and is expressed in all stages of terminal erythropoiesis. We evaluate plek2 mRNA and protein expression in sorted bone marrow erythroid precursors from WT, th1/th1, and transferrin-treated th1/th1 mice. Our data demonstrates a statistically significant increase in plek2 mRNA in th1/th1 relative to WT mice, with the highest expression of plek2 in poly-E, normalized in transferrin-treated th1/th1 mice (Figure 1A). A similar pattern of increased protein concentration in th1/th1 relative to WT mice and normalization in transferrin-treated th1/th1 mice is evident in sorted bone marrow samples (Figure 1B). Prior in vitro studies demonstrate that membrane localization of plek2 is required for erythroid differentiation. Thus, we performed sub-cellular fractionation in bone marrow erythroid precursors and determined for the first time that in sorted erythroblasts from WT bone marrow, plek2 is found exclusively in the cytoplasm in pro-E and in both cytoplasm and membrane from baso-E to ortho-E (Figure 2), co-localized with actin filaments in the membrane (data not shown). In contrast, sorted erythroblasts from th1/th1 bone marrow reveal membrane-associated plek2 starting from pro-E, demonstrating earlier co-localization with actin filaments (data not shown) and suggesting an earlier activation of plek2 and consequent actin cytoskeleton reorganization during erythroid differentiation in th1/th1 mice, normalized in transferrin-treated th1/th1 mice (Figure 2). Erythropoiesis involves a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, enucleation, and release into the circulation. For example, although Epo increases survival, Epo signaling also activates RacGTPases, inhibiting enucleation. Recent in vitro data demonstrates that knockdown of plek2 affected enucleation with significantly lower reticulocyte count. Although the involvement of RacGTPase in plek2-mediated erythroid differentiation has not been explored, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. Our data demonstrates that RacGTPase concentration is increased in sorted bone marrow erythroid precursors from th1/th1 relative to WT mice and normalized in transferrin-treated th1/th1 mice (Figure 1B). These results suggest that plek2 plays an important role in erythropoiesis likely as a key factor in the improved enucleation of transferrin-treated th1/th1 mice. Disclosures No relevant conflicts of interest to declare.

scholarly journals Treatment with at Homeopathic Complex Medication Modulates Mononuclear Bone Marrow Cell Differentiation

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Beatriz Cesar ◽  
Ana Paula R. Abud ◽  
Carolina C. de Oliveira ◽  
Francolino Cardoso ◽  
Raffaello Popa Di Bernardi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Marrow Cell ◽  
Macrophage Colony ◽  
Total Bone Marrow

A homeopathic complex medication (HCM), with immunomodulatory properties, is recommended for patients with depressed immune systems. Previous studies demonstrated that the medication induces an increase in leukocyte number. The bone marrow microenvironment is composed of growth factors, stromal cells, an extracellular matrix and progenitor cells that differentiate into mature blood cells. Mice were our biological model used in this research. We now reportin vivoimmunophenotyping of total bone marrow cells andex vivoeffects of the medication on mononuclear cell differentiation at different times. Cells were examined by light microscopy and cytokine levels were measuredin vitro. Afterin vivotreatment with HCM, a pool of cells from the new marrow microenvironment was analyzed by flow cytometry to detect any trend in cell alteration. The results showed decreases, mainly, in CD11b and TER-119 markers compared with controls. Mononuclear cells were used to analyze the effects ofex vivoHCM treatment and the number of cells showing ring nuclei, niche cells and activated macrophages increased in culture, even in the absence of macrophage colony-stimulating factor. Cytokines favoring stromal cell survival and differentiation in culture were inducedin vitro. Thus, we observe that HCM is immunomodulatory, either alone or in association with other products.

scholarly journals Establishment of a Spontaneous Stromal Microenvironment from Cord Blood Supports Human Dynamic Erythropoietic Temporal Maturation in Long-Term Serum- and Cytokine-Free 3D Cultures and Reveals a Distinct CD44hi Population

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2219-2219
Author(s):  
Susana Brito Dos Santos ◽  
Athanasios Mantalaris ◽  
Nicki Panoskaltsis
Keyword(s):  
Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Hla Typing ◽  
High Dose ◽  
Ex Vivo Model ◽  
Erythroid Progenitor ◽  
Erythroid Precursors

Dynamic cultures which can represent physiologic erythropoiesis in vitro require a three-dimensional (3D) architecture with a supportive microenvironment and addition of erythropoietin (EPO). We have previously reported on a 3D bone marrow (BM) biomimicry using polyurethane scaffolds to expand cord blood mononuclear cells (CBMNCs) in a serum- and cytokine-free environment, without addition of dexamethasone, for 28 days (D). CBMNCs were seeded (4x106 cells/scaffold), supplemented with 10ng/mL stem cell factor (SCF; D0-D28) and 100mU/mL EPO (D7-D28), with medium exchange every 3D and exposed to a hypoxia (5%)/normoxia (20%) schedule to mimic BM oxygen gradients. Hypoxia induced rapid erythroid commitment and established an early erythroid progenitor population in the absence of EPO. Normoxia and EPO was required at later maturational stages and enhanced the γ-globin to β-globin switch. We identified D7-D14 as crucial for endogenous cytokine production. Herein, we extended cultures to D48 using two high-dose EPO-stimulation cycles (1U/mL; D20 and D44) to enhance erythropoiesis and further define the microenvironment. Proliferation was higher after EPO pulses (p<0.05) but did not result in enhanced erythropoiesis, suggesting the absence of erythroid precursors. An allogeneic CB unit was added to "recharge" the cultured scaffolds at D39 and a new cycle of erythropoietic differentiation was initiated. Cell proliferation was 4.5-fold higher at D68, compared with that at D28. From D53-D68, CD71+CD235a+ cells were constantly produced (25-54%), corresponding with the presence of erythroid precursors supporting CFU-E and BFU-E. Erythroblastic islands were identified and maturing and enucleated reticulocytes/RBCs were abundant (19±2%; 1±0.3x106 cells) with expression of γ- and β-globin, band 3 and 4.1R RBC membrane proteins. To further evaluate the relative contributions of each CB unit to the "recharge" culture, seeded scaffolds were subjected to irradiation (or not) 48h prior to recharge. By D65, >30% of supernatant cells were CD71+/modCD235a+ and supported BFU-E and CFU-E. Proliferation in long-term cultures was attributed to the second CB unit, regardless of irradiation, as shown by HLA-typing of D68 cells; the first CBMNCs only contributed to establishment of the microenvironment. To further characterize erythroid differentiation dynamics, expression of CD44 vs CD235a was used to identify and sort three erythroid populations. Progenitors in CD44-/modCD235a- populations evolved to CD44modCD235amod, and then to CD44-/modCD235a+ cells, which constitute the most mature erythroid phenotype; CD44modCD235amod erythroid precursors supported mainly BFU-E and CFU-E. A unique CD44hiCD71mod population increased during culture, displayed myeloid progenitor morphology and only supported CFU-GM. This population did not express CD34, CD33 or CD14 but expressed c-KIT, which suggests a hematopoietic population that provides essential culture support. Further characterization of the spontaneously created microenvironment by in situ quantitative analysis of scaffold mid-sections during the 68-day culture showed varying and high dynamic expression of Nestin, STRO-1, CD146, CD68 and CD169 in separate cell populations as well as expression of RUNX2 and Osx. Osteopontin was not detected. In summary, a BM biomimicry composed of diverse stromal populations was spontaneously created in the 3D in vitro scaffold system. This microenvironment proved to effectively induce and sustain erythropoiesis to enucleation to at least D68 when supplied with a second source of CBMNCs, without addition of stroma-specific factors. A unique CD44hi immature monocyte/macrophage population was identified, which contributes to the inductive microenvironment, and distinct stages of human erythroid maturation could be identified using CD44 and CD235a. This work presents a novel and dynamic ex vivo model that can (1) recapitulate physiologic human erythropoiesis in steady-state and stress conditions, (2) capture the fetal to adult hemoglobin transition, (3) explore the direct role of oxygen on erythropoiesis, (4) assess the microenvironment relevant to erythropoiesis in the absence of serum and exogenous factors, (5) sustain long-term erythropoiesis with terminal maturation and, (6) explore the different stromal niche environments spontaneously created for the support of erythropoiesis. Disclosures Brito Dos Santos: GE Healthcare: Employment.

Aryl Hydrocarbon Receptor (AhR) Antagonist Stemregenin 1 (SR1) Enhances in Vitro- and in Vivo-Derived Platelets (PLTs) From Human Megakaryocytes (MKs)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3450-3450
Author(s):  
Yuhuan Wang ◽  
Vincent M. Hayes ◽  
Lin Lu ◽  
Xiaoji Chen ◽  
Rudy Fuentes ◽  
...  
Keyword(s):  
Cell Expansion ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Hematopoietic Progenitor Cell ◽  
Pulmonary Vasculature ◽  
Nsg Mice

Abstract Abstract 3450 Our goal is to generate sufficient PLTs from ex vivo-generated MKs for clinical utility in PLT transfusions. A critical step in this process begins with ex vivo-generated hMKs and deriving clinically relevant PLTs. We reported that infused mature, high-ploidy murine (m) MKs derived from fetal liver (FL) cells increased PLT counts in recipient mice in a clinically relevant fashion, thus avoiding the need to generate ex vivo functional PLTs. We examined whether this strategy applies to hMKs derived from FL cells (gestational age, 17–21 weeks) and bone marrow (BM) cells in a xeno-transfusion model using immunodeficient NOD/SCID/IL-2Rγcnull (NSG) mice. Infused hPLTs isolated from blood had a half-life (T1/2) of 10 hours (hrs), compared to 24 hrs for infused murine PLTs. The hPLTs were functional in NSG mice as demonstrated by their incorporation into growing thrombi in situ. Both hFL hematopoietic mononuclear cells and hBM-CD34+ cells were cultured in serum-free media supplemented with optimized cytokine cocktails to generate hMKs. In contrast to the murine studies where the FL cell-derived mMKs were the most efficient source of derived mPLTs, FL cell-derived hMKs had low ploidy (0% ≥ 8N ploidy), gave rise to ∼16 PLTs/infused hMK, and had a short T1/2 (6 hrs). In contrast, 17% of hBM cell-derived MKs had a ploidy of ≥ 8N, and after infusion into NSG mice, resulted in a wave of MKs transiently entrapped in the pulmonary microvasculature and then over ∼0.5–3 hrs released PLTs with a T1/2 of 10 hrs, comparable to infused hPLTs. Maximally, we achieved a level of 5% of circulating total PLTs being derived from human cells with ∼32 PLTs/infused hMK. These hPLTs were normal in size, displayed normal levels of surface markers, were functional, and incorporated into growing thrombi. One strategy to increase hPLT yield is to expose developing MKs to drugs reported to increase MK maturation, thrombopoiesis, and/or facilitate hematopoietic progenitor cell expansion. Such drugs include dimethylfasudil (diMF) (an inhibitor of several kinases involved in polyploidization), UNC0638 (a G9a histone methyltransferase inhibitor), SR1 (an AhR antagonist), and nicotinamide (a sirtuin histone/protein deacetylases inhibitor). Although diMF promoted size and polyploidization of hMKs, diMF markedly worsened yield of PLTs/infused hMK and decreased PLTs T1/2 in vivo. UNC0638 led to significant cell expansion, but lowered hMKs ploidy and PLTs/infused hMK yield. Nicotinamide increased maturation, size and polyploidization of hMKs, but PLT release following MK infusion needs further study. Of note, SR1 that has been reported to promote the expansion of human HSC, not only increased size and ploidy of hMKs, but also hPLT release in vitro and in vivo. SR1-treated hMKs resulted in a 3-fold increased yield of normal size, T1/2 and functional PLTs/infused hMK compared to a DMSO-treated control. In summary, like mMKs, infused hMKs into mice release PLTs in the pulmonary vasculature though at a lower efficiency. Released hPLTs were functional and T1/2 was as expected. diMF enhanced MK ploidy, but worsened PLT yield and T1/2, while an AhR antagonist SR1 that also improved MK ploidy appears to markedly enhance yield of PLT/infused hMK, while maintaining T1/2. The ability of SR1 to enhance PLT release from induced pluripotent stem cells (iPSCs)-derived MKs remains to be tested, but this drug appears to be a strong candidate for a therapeutic strategy to take ex vivo-grown hMKs and generate PLTs in clinical relevant numbers. Disclosures: No relevant conflicts of interest to declare.

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