CFU-E Research Articles

Decreased "ineffective erythropoiesis" preserves polycythemia in mice under long-term hypoxia.

Hypoxia induces innumerable changes in humans and other animals, including an increase in peripheral red blood cells (polycythemia) caused by the activation of erythropoiesis mediated by increased erythropoietin (EPO) production. However, the elevation of EPO is limited and levels return to normal ranges under normoxia within 5-7 days of exposure to hypoxia, whereas polycythemia continues for as long as hypoxia persists. We investigated erythropoiesis in bone marrow and spleens from mouse models of long-term normobaric hypoxia (10 % O2) to clarify the mechanism of prolonged polycythemia in chronic hypoxia. The numbers of erythroid colony-forming units (CFU-E) in the spleen remarkably increased along with elevated serum EPO levels indicating the activation of erythropoiesis during the first 7 days of hypoxia. After 14 days of hypoxia, the numbers of CFU-E returned to normoxic levels, whereas polycythemia persisted for >140 days. Flow cytometry revealed a prolonged increase in the numbers of TER119-positive cells (erythroid cells derived from pro-erythroblasts through mature erythrocyte stages), especially the TER119 (high) CD71 (high) population, in bone marrow. The numbers of annexin-V-positive cells among the TER119-positive cells particularly declined under chronic hypoxia, suggesting that the numbers of apoptotic cells decrease during erythroid cell maturation. Furthermore, RT-PCR analysis showed that the RNA expression of BMP-4 and stem cell factor that reduces apoptotic changes during erythroid cell proliferation and maturation was increased in bone marrow under hypoxia. These findings indicated that decreased apoptosis of erythroid cells during erythropoiesis contributes to polycythemia in mice during chronic exposure to long-term hypoxia.

Do two different stem cell grafts: G-CSF stimulated and unstimulated bone marrow differ according to hematopoietic colony forming capacity?

The study was designed to compare colony forming capacity of granulocyte-colony stimulating factor (G-CSF) stimulated bone marrow (G-BM) with standard unstimulated bone marrow (U-BM) of healthy donors of pediatric patients. CFU-Assay results of 26 healthy donors of pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) were analyzed retrospectively. 13 of donors received 10 μg/kg per day of G-CSF as a single injection for 3 consecutive days and other 13 of donors had unstimulated BM. Colony forming capacity of hematopoietic stem cells evaluated with Colony Forming Unit-Assay (CFU-Assay) with in semi-solid agar culture medium after 14-18 days of culture period. CFU-Assay results of G-BM and U-BM (expressed as means) were; Burst Forming Unit-Erythroid (BFU-E): 15.20 × 10(4)/kg and 8.38 × 10(4)/kg, Colony Forming Unit-Granulocyte Macrophage (CFU-GM): 10.35 × 10(4)/kg and 5.67 × 10(4)/kg, Colony Forming Unit-Erythroid (CFU-E): 0.59 × 10(4)/kg and 0.33 × 10(4)/kg, CFU-Granulocyte Erythroid Macrophage Megakaryocyte (CFU-GEMM): 0.52 × 10(4)/kg and 0.53 × 10(4)/kg respectively. BFU-E and CFU-GM capacity of G-BM was increased and statistically significantly different than standard U-BM (p ⩽ 0.01). In conclusion, increased colony forming capacity of hematopoietic stem cells of G-BM when compared with standard unstimulated BM could be a major advantage for transplantation.

Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis

Key transcriptional regulators of terminal erythropoiesis, such as GATA-binding factor 1 (GATA1) and T-cell acute lymphocytic leukemia protein 1 (TAL1), have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here, we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor transcription factor Dp-2 (Tfdp2) were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression and knockdown of Tfdp2 results in significantly reduced rates of proliferation as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis.

Transfusion begets anemia

Following trauma, transfusion of aged stored blood is often necessary yet associated with increased morbidity and mortality. Despite blood replacement, many patients have a prolonged anemia requiring further transfusions. The effects of aged blood on bone marrow (BM) hematopoiesis have not been studied, and we hypothesized that stored blood suppresses BM function. Blood from Sprague-Dawley rats was stored for 1, 14, or 28 days with the industry preservative citrate-phosphate-dextrose-adenine-1 (CPDA-1). For in vitro studies, 5% supernatant was incubated with normal rat BM and cultured for erythroid (CFU-E) and granulocyte-macrophage (CFU-GM) colony-forming units. Data were compared with cultures of BM alone, 5% control plasma (negative control), and 12% CPDA-1. For in vivo studies, rats were transfused with stored supernatants (5% estimated blood volume (EBV) over 30 minutes). BM from each recipient was cultured for CFU-E and CFU-GM at 3 hours after transfusion. Data were compared with cultures of BM alone. Difference between groups determined by analysis of variance and Tukey's multiple comparison test. In vitro exposure to CPDA-1, control plasma, or 1-day supernatant (D1) had no effect on BM growth compared with BM alone. In vitro exposure to 14-day (D14) and 28-day (D28) supernatant significantly suppressed CFU-E by 60% and CFU-GM growth by 71% (both p < 0.05) compared with D1 or medial alone. There were no differences between D14 and D28. In vivo exposure to D14 reduced BM CFU-E and CFU-GM growth by 55% (both p < 0.05) compared with D1 supernatant. Plasma from aged blood adversely affects CFU-E and CFU-GM growth in rats. The effect is not mediated by CPDA-1. Transfusion of aged stored blood may contribute to BM dysfunction in critically ill patients, resulting in persistent anemia and the need for further transfusion. This BM dysfunction may also partly explain the observed increased susceptibility to infection.

Dynamic Changes Of DNA Methylation and a Functional Role For TET2 DNA Dioxygenase In Human Erythroid Differentiation

Erythropoiesis is a process by which multipotent hematopoietic stem cells proliferate, differentiate and eventually form mature erythrocytes. This process contains eight distinct differentiation stages including burst-forming unit-erythroid (BFU-E), colony-forming unit-erythroid (CFU-E), proerythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, reticulocyte and mature erythrocyte. Unlike most cell types, an important feature of erythropoiesis is that following each of the three or four mitoses that occur during terminal erythroid differentiation, the daughter cells are distinctly different from the parent cell from which they are derived. Thus, erythropoiesis is a complex process that requires tight regulation. The most extensively studied regulators of erythroid differentiation include the EPO/EPOR system and two major transcription factors, GATA1 and KLF1. In contrast to the well-established roles of growth factors, cytokines and transcription factors in regulating erythropoiesis, the regulation of erythropoiesis by other mechanisms is much less understood. In the present study, we explore the changes in DNA methylation during human terminal erythroid differentiation and DNA methylation/demethylation in human erythropoiesis.The methylation status of DNA influences many biologic processes. It has been recently reported that global demethylation occurs during both murine and human erythropoiesis. However, the dynamics of DNA methylation changes, the underlying molecular mechanism(s), and the function of DNA demethylation in erythropoiesis are not clear. To address these issues, we performed next-generation bisulfite sequencing on highly purified human erythroblasts at distinct differentiation stages. We show that while there is a global hypomethylation as terminal erythropoiesis proceeds, stage-specific analysis revealed that a significant proportion of differential methylation includes gains of methylation. Moreover, genes that presented with DNA methylation changes could be categorized into 3 groups based on the dynamics of their methylation changes. As Ten-eleven-translocation proteins (TETs) have been implicated in DNA demethylation by converting 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc), we attempted to explore the role of TETs in DNA demethylation and terminal erythroid differentiation. We show that 5hmc is progressively increased during human terminal erythroid differentiation. Importantly, knockdown of TET2 by shRNA in human CD34+ cells impaired the production of 5hmc as well as terminal erythroid differentiation. Our findings demonstrate the complexity of DNA methylation dynamics and identify a functional role for TET2 in human erythroid differentiation. These findings provide new and novel insights into the mechanistic understanding of normal and disordered erythropoiesis. As aberrant DNA methylation underlies many hematological diseases including the dyserythropoiesis of myelodysplastic syndromes, we suggest that these finding also provide novel insights into these diseases. Disclosures:No relevant conflicts of interest to declare.

The Erythropoietin Receptor Regulates The Number Of Cell Divisions and The Duration Of Erythroblast Terminal Differentiation By Regulating Erythroblast Iron

Signaling by the erythropoietin receptor (EpoR) is essential for the survival of definitive colony-forming unit-erythroid (CFU-e) progenitors and their erythroblast progeny. Here we used EpoR-null embryos to ask whether EpoR signaling might also exert essential non-survival functions in erythropoiesis.To address this, we rescued EpoR-null fetal liver cells from death by transducing them in vitro with either the anti-apoptotic protein Bcl-xL, or, as control, with the wild-type EpoR. The Bcl-xL-transduced EpoR-null cells survived, expressed hemoglobin and underwent morphological erythroid maturation and enucleation. However, unlike exogenous EpoR, exogenous Bcl-xL was unable to support the formation of EpoR-null CFU-e colonies in methylcellulose. The absence of colonies was explained by the finding that the Bcl-xL-transduced progenitors underwent fewer cell divisions than equivalent EpoR-transduced cells (1.1 vs. 2.9 in 24 hr, respectively) and had a slower rate of intra-S phase DNA synthesis, suggesting longer S phase duration. Multispectral imaging showed that the Bcl-xL-transduced cells matured prematurely, attaining smaller cell and nuclear size and a lower nuclear/cytoplasmic ratio at earlier time points than EpoR-transduced cells. Premature maturation was also evident by flow cytometric analysis. Thus, EpoR-null fetal liver cells in vivo arrest in their differentiation at the transition from subset S0 (Ter119-neg CD71-low) to S1 (Ter119-neg CD71-high) (Pop et al, PLoS Biology 2010). Rescue with EpoR in vitro allows EpoR-null progenitors to resume differentiation, sequentially upregulating CD71 and Ter119. By contrast, rescue of EpoR-null cells with Bcl-xL results in their premature upregulation of Ter119 and failure to upregulate CD71 to high levels. The cell cycle and differentiation deficits in Bcl-xL-supported, EpoR-null erythropoiesis were associated with a slower loss of DNA methylation from the erythroid genome, and with slower erythroid gene transcription.CD71 (the transferrin receptor) is a known target of EpoR and Stat5 signaling. We asked whether the deficits of EpoR-null erythropoiesis might be the result of low cell surface CD71 and the consequent reduced iron transport. In support of this hypothesis, we found that EpoR-null fetal liver cells that are transduced with both CD71 and Bcl-xL resume the normal maturation rate characteristic of EpoR-supported differentiation, as judged by multispectral imaging measurements of cell size and nuclear/cytoplasmic ratio. Further, we were able to restore rapid S phase to Bcl-xL-transduced EpoR-/- erythroblasts by culturing them in the presence of the cell-permeant iron chelator Fe-SIH (salicylaldehyde isonicotinoyl hydrazone), which is able to supply the cell interior with iron even in the absence of CD71 (Figure 1). We suggest that EpoR-mediated upregulation of CD71 at the onset of erythroid terminal differentiation determines the number and duration of erythroblast cell divisions by regulating iron homeostasis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Inducing Definitive Erythropoiesis From Human Embryonic Stem Cells Through a Novel Intracellular MPL Dimerization Strategy

ObjectivesUnlimited 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 and fetal rather than adult hemoglobins. 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. MethodsHuman 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. ResultsF36V-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. 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=0.006; 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. Globin analysis by HPLC demonstrated that although no detectable beta-globin expression was observed with EPO, CID treatment induced the presence of beta-globin and increased the gamma: epsilon globin ratio, suggesting a shift toward definitive erythropoiesis. Signaling studies 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=0.001; n=4) indicating that MPL dimerization induced erythropoiesis is dependent on AKT signaling. Gene expression analysis by qPCR indicated that MPL dimerization upregulates a network of genes (downstream of AKT signaling) associated with the regulation of cell cycle, apoptosis, and erythroid differentiation, including GATA1, CDKN1A, RB1, VEGFA, and BCL-xL with a corresponding reduction in both apoptosis and cell cycle progression. ConclusionIn summary, we have identified a novel EPO-independent approach that is not only more efficient at erythropoiesis but is also able to augment EPO induced erythropoiesis. The mechanistic insights gained from this study opens up potentially new approaches toward the generation of therapeutically relevant quantities of RBCs for transfusion. Disclosures:No relevant conflicts of interest to declare.

Noonan Syndrome Peripheral Blood Cells Show Enhanced Spontaneous Colonies Growth In Vitro and Hyperactive JAK and RAS Intracellular Signalling Pathways

Noonan Syndrome (NS) is an autosomal dominant congenital disorder characterized by several defects at births. In addition, children with NS show splenomegaly and monocytosis, and are predisposed to develop myelo proliferative disorders (MPD) which may resolve spontaneously or progress in an aggressive clinical course similar to Juvenile Myelomonocytic Leukemia (JMML). Somatic PTPN11 mutations, which encodes the protein tyrosine phosphatase Shp2, affect around 35% of patients with JMML, while germline PTPN11 mutations cause around 50% of NS cases (Cratz et al. Blood, 2005). Moreover, mutations in other genes affecting the RAS/RAF/ERK pathway, such as SOS1 and KRAS, have been identified in PTPN11-negative NS patients (Tartaglia et al. Nature Genetics, 2007). Yet, both JMML and NS/MPD patients show hypersensitivity to GM-CSF and spontaneous colonies growth (Lavin et al. Pediatric Blood Cancer, 2008). These evidences prompted the hypothesis that children with NS could maintain an abnormal status of hematopoiesis affecting in particular the myelomonocytic lineage.We studied 23 children with NS and 16 age-matched children with other genetic defect, affecting neither JAK-STAT nor RAS/RAF/ERK pathways. Peripheral blood (PB) cells were cultured in vitro in order to evaluate the spontaneous and factor-stimulated growth of colony-forming units. In addition, PB monocytes were evaluated for IL-6 (50 ng/mL)-induced response of pSTAT3, and GM-CSF-induced response of pSTAT5 and pERK (10 and 0.1 ng/mL respectively), measured by phosphoflow technique, as previously described (Gaipa et al, Leukemia, 2008).We observed a significantly enhanced in vitro spontaneous growth of erythroid colony-forming units (BFU-E) in NS (n=15) as compared to control subjects (n=10) (mean ± SD: 79.67% ± 30.15% vs 22.60% ± 23.00%, respectively; p=0.0004), and an augmented growth of granulocyte-macrophage units (CFU-GM), although not significantly (19.13% ± 17.20% vs 10.90% ± 17.72%; p>0.05). This enhanced capacity of NS cells to promote colony formation was confirmed also in the presence of growth factors in the medium.Phosphoflow assay showed an enhanced amount of stimulated pSTAT3 and pSTAT5 positive cells in NS patients as compared to control subjects (pSTAT3: 41.01% ± 32.97% (n=18) vs 22.99% ± 22.38% (n=13); p=ns, and pSTAT5: 98.20% ± 2.55% (n=5) vs 63.40% ± 40.17% (n=5), p<0.05), Interestingly, preliminary experiments showed that NS patients also have higher rate of pERK response upon stimulation with GM-CSF.In summary, we demonstrated that children with NS, but without MPD, do have significantly enhanced capacity of both BFU-E and CFU-GM units formation in vitro. GM-CSF hypersensitivity has been demonstrated in NS/MPD (Lavin et al. Pediatric Blood Cancer, 2008), thus supporting the observed enhanced growth of CFU-GM units in NS patients with persistent monocytosis. Intriguingly, abnormal growth of BFU-E was not previously reported in these patients. The biological significance of these enhanced growth capacities, particularly in absence of recognized MPD status, should be further investigated. Signalling in transduction pathways affected by Shp2 protein seems to be latently activated in NS, although these observations need to be fully validated with further experiments. Collectively, our findings open new opportunities to investigate the almost unknown hematological alterations existing in NS patients. In addition, they may lead to an enrichment of the laboratory tools potentially able to recognize signs of progression toward malignant MPD (including JMML) in children with NS. Disclosures:No relevant conflicts of interest to declare.

Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid terminal differentiation in the fetal liver.

Tropomodulin (Tmod) is a protein that binds and caps the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse tropomodulin3 (Tmod3) leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. Erythroid burst-forming unit and colony-forming unit numbers are greatly reduced, indicating defects in progenitor populations. Flow cytometry of fetal liver erythroblasts shows that late-stage populations are also decreased, including reduced percentages of enucleated cells. Annexin V staining indicates increased apoptosis of Tmod3(-/-) erythroblasts, and cell-cycle analysis reveals that there are more Ter119(hi) cells in S-phase in Tmod3(-/-) embryos. Notably, enucleating Tmod3(-/-) erythroblasts are still in the process of proliferation, suggesting impaired cell-cycle exit during terminal differentiation. Tmod3(-/-) late erythroblasts often exhibit multilobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3(-/-) fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell-cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation.

PRDM11 is dispensable for the maintenance and function of hematopoietic stem and progenitor cells

Hematopoietic stem cells (HSC)11Hematopoietic stem cell (HSC), hematopoietic stem and progenitor cell (HSPC), bone marrow (BM), bone marrow transplantation (BMT), acute myeloid leukemia (AML), peripheral blood (PB), multipotent progenitor (MPP), pre-megakaryocyte/erythroid (preMegE), megakaryocytic progenitor (MkP), pre-granulocyte/macrophage (preGM), granulocyte/macrophage progenitors (GMP), common lymphoid progenitors (CLP), colony forming unit erythroid (CFU-E), proErythroid (proE), colony forming unit megakaryocyte (CFU-Mk), colony forming unit granulocyte macrophage (CFU-GM), megakaryocyte (Mk), LSK (Lineage−, Sca1+, c-Kithi). supply organisms with life-long output of mature blood cells. To do so, the HSC pool size has to be maintained by HSC self-renewing divisions. PRDM3 and PRDM16 have been documented to regulate HSC self-renewal, maintenance and function. We found Prdm11 to have similar expression patterns in the hematopoietic stem and progenitor cell (HSPC) compartments as Prdm3 and Prdm16. Therefore, we undertook experiments to test if PRDM11 regulates HSC self-renewal, maintenance and function by investigating the Prdm11−/− mice. Our data shows that phenotypic HSPCs are intact in bone marrow (BM) of one-year-old Prdm11−/− mice. In addition, Prdm11−/− mice were able to fully regenerate the hematopoietic system upon BM transplantation (BMT) into lethally irradiated mice with a mild drop in lymphoid output only. Taken together, this suggests that PRDM11, in contrast to PRDM3 and PRDM16, is not directly involved in regulation of HSPCs in mice.

ZFP36L2 is required for self-renewal of early burst-forming unit erythroid progenitors

Stem cells and progenitors in many lineages undergo self- renewing divisions, but the extracellular and intracellular proteins that regulate this process are largely unknown. Glucocorticoids stimulate red cell formation by promoting self-renewal of early erythroid burst forming unit-erythrocyte (BFU-E) progenitors1-4. Here we show that the RNA binding protein Zfp36l2 is a transcriptional target of the glucocorticoid receptor (GR) in BFU-Es and is required for BFU-E self-renewal. Zfp36l2 is normally downregulated during erythroid differentiation from the BFU-E stage but its expression is maintained by all tested GR agonists that stimulate BFU-E self-renewal, and the GR binds to several potential enhancer regions of Zfp36l2. Knockdown of Zfp36l2 in cultured BFU-E cells did not affect the rate of cell division but disrupted glucocorticoid-induced BFU-E self-renewal, and knockdown of Zfp36l2 in transplanted erythroid progenitors prevented expansion of erythroid lineage progenitors normally seen following induction of anemia by phenylhydrazine treatment. Zfp36l2 preferentially binds to mRNAs that are induced or maintained at high expression levels during terminal erythroid differentiation and negatively regulates their expression levels. Thus Zfp36l2 functions as part of molecular switch promoting BFU-E self-renewal and thus a subsequent increase in the total numbers of CFU-E progenitors and erythroid cells that are generated.

Biochemical measurements on single erythroid progenitor cells shed light on the combinatorial regulation of red blood cell production

Adult bone marrow (BM) erythrocyte colony-forming units (CFU-Es) are important cellular targets for the treatment of anemia and also for the manufacture of red blood cells (RBCs) ex vivo. We obtained quantitative biochemical measurements from single and small numbers of CFU-Es by isolating and analyzing c-Kit(+)CD71(high)Ter119(-) cells from adult mouse BM and this allowed us to identify two mechanisms that can be manipulated to increase RBC production. As expected, maximum RBC output was obtained when CFU-Es were stimulated with a combination of Stem Cell Factor (SCF) and Erythropoietin (EPO) mainly because SCF supports a transient CFU-E expansion and EPO promotes the survival and terminal differentiation of erythroid progenitors. However, we found that one of the main factors limiting the output in RBCs was that EPO induces a downregulation of c-Kit expression which limits the transient expansion of CFU-Es. In the presence of SCF, the EPO-mediated downregulation of c-Kit on CFU-Es is delayed but still significant. Moreover, treatment of CFU-Es with 1-Naphthyl PP1 could partially inhibit the downregulation of c-Kit induced by EPO, suggesting that this process is dependent on a Src family kinase, v-Src and/or c-Fyn. We also found that CFU-E survival and proliferation was dependent on the level of time-integrated extracellular-regulated kinase (ERK) activation in these cells, all of which could be significantly increased when SCF and EPO were combined with mouse fetal liver-derived factors. Taken together, these results suggest two novel molecular strategies to increase RBC production and regeneration.

Inhibition of the CRBN-DDB1-CUL4-ROC1 E3 Ubiquitin Ligase Mediates the Anti-Proliferative and Immunomodulatory Properties of Lenalidomide

Abstract 919Lenalidomide is a highly effective drug for the treatment of del(5q) MDS and multiple myeloma, and its use in a range of other conditions is being actively explored. Despite its increasing use for the treatment of malignancies, the precise mechanism of action of lenalidomide has not been established. We sought to identify the direct protein targets of lenalidomide using a quantitative, mass spectrometry-based proteomic approach we developed.Using a validated derivative of lenalidomide immobilized to beads, we identified DDB1 as a target of the drug by affinity enrichment of protein binders and analysis by high performance LC-MS/MS. DDB1, together with CRBN, CUL4A, and ROC1, forms an E3 ubiquitin ligase known as CRBN-CRL4. We confirmed that members of the complex bind to the immobilized lenalidomide derivative, and could be competed off with soluble lenalidomide, further supporting the role of CRBN-CRL4 in the actions of lenalidomide.CRL4 targets multiple proteins for ubiquitination and subsequent proteasomal degradation, including the cell cycle regulators CDKN1A (p21) and CDKN1B (p27), as well as the DNA licensing factor CDT1. We hypothesized that lenalidomide disrupts the ubiquitination of these and other proteins, leading to increased levels of the respective targets. We found that treatment of the lenalidomide sensitive cell line MM1S and NCI-H929 increased protein levels of p21, p27 and CDT1 in a dose and time dependent manner. Furthermore, overexpression of these three targets led to growth inhibition. Similarly, knockdown of DDB1, CUL4A, ROC1 and CRBN by lentiviral shRNAs increased p21 and p27 protein levels and inhibited growth of these cell lines.Lenalidomide is also known to increase IL-2, promote erythropoiesis and inhibit TNF-alpha. We found that in activated primary human T cells, shRNA knockdown of DDB1 recapitulated the stimulatory effects of lenalidomide on IL-2 expression levels and release. We also found that shRNA knockdown of DDB1 and CRBN recapitulated the pro-erythropoietic effects of the drug with an increase in the number of colony-forming units-erythroid (CFU-E) compared to control knockdown. Experiments studying the effects on TNF-alpha are underway.To further establish that the CRBN-CRL4 complex is the target of lenalidomide, we tested a previously published mutant form of CRBN which prevents binding of the drug to the complex. Ectopic expression of this mutant CRBN conferred resistance to lenalidomide induced cell death to multiple myeloma cells. It also resulted in the loss of CFU-E production by lenalidomide.To gain further insight into how lenalidomide might disrupt the function of the CRBN-CRL4 complex, we did immunoprecipitation against CRBN with or without the drug and found that lenalidomide disrupts the formation of the complex by preventing binding of ROC1, the adaptor protein to ubiquitin charged E2 conjugating enzyme. Using in vivo and in vitro ubiquitination assays, we also demonstrated that lenalidomide inhibits the auto-ubiquitination of CRBN. We are currently performing a ubiquitin profiling experiment to identify other protein targets that might be affected by the disruption of the CRBN-CRL4 complex by lenalidomide.Our study establishes that lenalidomide's antiproliferative and immunomodulatory properties rely on binding to CRBN-CRL4 and inhibiting its function as ubiquitin ligase. Ito et al. showed 2010 that the same mechanism is also responsible for the teratogenic effects of thalidomide. The characterization of lenalidomide as a specific E3 ubiquitin ligase inhibitor will provide insight into the mechanism of therapeutic efficacy in MDS and multiple myeloma, and serves as a proof-of-concept that selective ubiquitin ligases are efficacious targets for cancer therapy. Disclosures:No relevant conflicts of interest to declare.

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

AbstractAbstract 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.

Role of bone marrow and mesenchymal stem cells in healing after traumatic injury

The role of bone marrow-derived cells (BMDCs) and mesenchymal stem cells (MSC) in healing of traumatic-induced injury remains poorly understood. Mesenteric lymph duct ligation (LDL) results in decreased BMDC mobilization and impaired healing. We hypothesized that LDL-mediated impaired healing would be abrogated by reinjection of BMDC or MSC. Sprague-Dawley rats were subjected to LDL + lung contusion (LC+LDL) with or without injection of BMDCs or MSCs. Unmanipulated control (UC) and lung contusion alone (LC) served as controls. BMDC and MSC homing was assessed by hematopoietic progenitor cell (HPC [granulocyte-, erythrocyte-, monocyte-, and megakaryocyte colony-forming units; erythroid burst-forming units; and erythroid colony-forming units]) colony growth and immunofluorescent microscopic tracking of tagged MSC, respectively. Histologic lung injury score (LIS) was used to grade injury. Data are mean ± SD. *P < .05/Student t test. Lung HPC growth was decreased in LC+LDL versus LC alone (HPC colonies: 2 ± 2, 4 ± 3, 4 ± 2 vs. 11 ± 2, 20 ± 6, 22 ± 9. *P < .05). LC+LDL had greater degree of lung injury on days 5 and 7 LC alone (LIS: 5 ± 1, 4 ± 1 vs. 3 ± 1, 1 ± 0.4. *P < .05). BMDC injection into rats with LC + LDL increased lung HPC growth to LC level (HPC colonies: 12 ± 2, 19 ± 5, 17 ± 4 vs 11 ± 2, 20 ± 6, 22 ± 9. P > .05). Injected MSCs into LC+LDL rats homed preferentially to contused versus noncontused lung (MSC/high-powered field: 6 ± 4 vs. 2 ± 2 *P < .05). Either BMDC or MSC injection into LC+LDL rats returned lung injury to LC level on day 7 (LIS: 1 ± 0.4 and 1 ± 1 vs. 1 ± 0.4. P > .05). LDL-mediated impaired tissue healing is abrogated by either whole BMDC or MSC injection. This highlights the critical role of BMDC and MSC on healing of trauma-induced injury.

Low-intensity pulsed ultrasound-mediated stimulation of hematopoietic stem/progenitor cell viability, proliferation and differentiation in vitro

Low-intensity pulsed ultrasound (LIPUS) stimulated the viability, proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) from fresh and cryopreserved peripheral blood leukapheresis product, as well as cord blood when applied for 10 min each day for 4 days. Cell viability, proliferation and differentiation were assessed on day 5 by viable cell counting, MTS proliferation assay, flow cytometry, and colony-forming unit assay. LIPUS stimulation: (i) enhanced the proliferation of fresh HSPC and maintained the viability of cryopreserved HSPC in vitro; (ii) did not affect the percentage of CD34(+) and CD14(+) cells; and (iii) enhanced burst-forming unit-erythroid colony formation. Hence, we suggest that this novel LIPUS stimulation approach might enhance the efficacy of clinical transplantation and cellular therapies using HSPC.

SPARC promotes the development of erythroid progenitors

Secreted protein acidic and rich in cysteine (SPARC) is a well-recognized regulator that affects cell proliferation, differentiation, and survival. Here, we investigated the impact of SPARC on erythropoiesis. Erythropoiesis in bone marrow (BM) from SPARC(-/-) mice was analyzed by flow cytometry and colony-forming assays. The mechanisms that affected erythropoiesis were investigated by BM transplantation experiments. CD34(+) cells from umbilical cord blood were isolated by MiniMACS, and the effect of SPARC on human erythropoiesis was assessed by colony-formation assay and erythroid differentiation culture. The hemoglobin level in peripheral blood (PB) was lower in SPARC(-/-) mice. Neither red blood cell count in PB nor the percentage of Ter119(+) erythrocytes in BM showed significant difference between SPARC(-/-) and WT mice. However, the ability of SPARC(-/-) bone marrow cells (BMCs) to form erythroid burst-forming units (BFU-E), as well as spleen colony-forming units (CFU-S8), was significantly decreased. The addition of exogenous SPARC could promote the formation of BFU-E from SPARC(-/-) BMCs in vitro. And the impaired ability of SPARC(-/-) BMCs to form BFU-E could be restored by transplanted into WT BM microenvironment, whereas the BFU-E formation of WT BMCs was impaired after transplanted into SPARC(-/-) recipients. Furthermore, exogenous SPARC promoted umbilical cord blood CD34(+) cells to form erythroid colonies and the hemoglobinization of erythroid, but did not affect the expression levels of glycophorin A and CD71. In conclusion, our results indicate that SPARC promotes the development of erythroid progenitors, but does not affect terminal erythroid differentiation.

Measurement of generation‐dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture.

Enucleation of human erythroblasts involves non-muscle myosin IIB

AbstractMammalian erythroblasts undergo enucleation, a process thought to be similar to cytokinesis. Although an assemblage of actin, non-muscle myosin II, and several other proteins is crucial for proper cytokinesis, the role of non-muscle myosin II in enucleation remains unclear. In this study, we investigated the effect of various cell-division inhibitors on cytokinesis and enucleation. For this purpose, we used human colony-forming unit-erythroid (CFU-E) and mature erythroblasts generated from purified CD34+ cells as target cells for cytokinesis and enucleation assay, respectively. Here we show that the inhibition of myosin by blebbistatin, an inhibitor of non-muscle myosin II ATPase, blocks both cell division and enucleation, which suggests that non-muscle myosin II plays an essential role not only in cytokinesis but also in enucleation. When the function of non-muscle myosin heavy chain (NMHC) IIA or IIB was inhibited by an exogenous expression of myosin rod fragment, myosin IIA or IIB, each rod fragment blocked the proliferation of CFU-E but only the rod fragment for IIB inhibited the enucleation of mature erythroblasts. These data indicate that NMHC IIB among the isoforms is involved in the enucleation of human erythroblasts.

Effects of oxidative stress on hematopoiesis of hematopoietic stem and progenitor cells with iron overload

To establish a model of hematopoietic stem and progenitor cells with iron overload derived from umbilical cord blood (UCB) cells and explore the effects of reactive oxygen species (ROS) on the hematopoiesis of hematopoietic stem and progenitor cells with iron overload. The model was established by adding different concentrations (50, 100, 200, 400 µmol/L)of ferric citrate (FAC) into mononuclear cells from UCB and culturing for different times (6, 12, 24 h). The UCB cells were divided into 4 groups: control group, group FAC, group FAC+N-acetyl-L-cysteine (NAC) and group FAC+ L-Glutathione (GSH). Then the changes of ROS, labile iron pool (LIP), apoptosis, the capacity of hematopoietic colony forming (CFU-E, BFU-E, CFU-GM, CFU-mix) and the percentage and the numbers of CD34(+), CD33(+), GlyA(+) cells were detected. And the changes of these indices were tested after the treatment of iron overload UCB with antioxidants (NAC and GSH). UCB cells were cultured with the addition of FAC at different concentrations for different times. The level of total ROS increased in time and concentration-dependent manners. The intracellular level of ROS peaked when cultured at 200 µmol/L of FAC for 24 hours. Cells were treated with antioxidants NAC or GSH after cultured with 200 µmol/L FAC for 24 hours. Then the ROS levels of total cells, myeloid cells and erythroid cells decreased markedly versus normal controls. The LIP of total cells, myeloid cells and erythroid cells increased markedly when cells were cultured at 200 µmol/L of FAC for 24 hours versus normal controls (P < 0.05). NAC and GSH had no effect on the level of LIP. The apoptotic rates of FAC-treated cells [(20.90 ± 3.45)%] increased significantly versus normal controls [(9.20 ± 1.29)%] (P < 0.05). The capacity of hematopoietic colony forming in FAC treated cells decreased markedly versus normal controls. The percentage and numbers of CD34(+), CD33(+), GlyA(+) cells of FAC-treated cells also decreased significantly versus normal controls (P < 0.05). And these changes could be recovered by the addition of NAC or GSH. Oxidative stress plays an important role in the injuries of hematopoiesis of hematopoietic stem and progenitor cells with iron overload by inducing the generation of ROS. These findings may help us find a specific target and improve the therapeutic efficacy of ineffective hematopoiesis in patients with iron overload.