ALTERATIONS IN POLYRIBOSOMES DURING ERYTHROID CELL MATURATION

This communication presents a morphological study of the changes in ribosome content and organization which occur during the maturation of erythroid cells of the phenylhydrazine-treated rabbit. Electron micrographs of thin sectioned nucleated and non-nucleated erythroid cells have been subjected to a quantitative analysis of the distribution of ribosomes as polyribosomes of various sizes and as single ribosomes. The ribosomes of nucleated erythroid cells of marrow are virtually all arranged in the polyribosome configuration consisting of clusters of 2 to 6 individual ribosomes. These cells are the most active in the erythroid series in protein biosynthesis. During maturation to the non-nucleated reticulocyte stage, found in the circulating blood, there is a decrease in protein synthesizing capacity, a fall in total ribosome content, and, more significantly, a decrease in the number and size of polyribosomes. Maturation to the ribosome-free erythrocyte, either under in vitro or in vivo conditions, entails a further decrease in protein synthesis which correlates with a progressive disaggregation of the biosynthetically active polyribosomes into smaller clusters and inactive single ribosomes. Possible models which may account for the stability of the polyribosome and for the mechanism of polyribosome dissociation are discussed.

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Protein synthesis is required for rapid degradation of tubulin mRNA and other deflagellation-induced RNAs in Chlamydomonas reinhardi

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.54-61.1986 ◽

1986 ◽

Vol 6 (1) ◽

pp. 54-61

Author(s):

E J Baker ◽

L R Keller ◽

J A Schloss ◽

J L Rosenbaum

Keyword(s):

Protein Synthesis ◽

Transcriptional Control ◽

Protein S ◽

Protein Synthesis Inhibitors ◽

Control Factors ◽

Flagellar Proteins ◽

The Stability ◽

And Control

After flagellar detachment in Chlamydomonas reinhardi, there is a rapid synthesis and accumulation of mRNAs for tubulin and other flagellar proteins. Maximum levels of these mRNAs (flagellar RNAs) are reached within 1 h after deflagellation, after which they are rapidly degraded to their predeflagellation levels. The degradation of alpha- and beta-tubulin RNAs was shown to be due to the shortening of their half-lives after accumulation (Baker et al., J. Cell Biol. 99:2074-2081, 1984). Deflagellation in the presence of protein synthesis inhibitors results in the accumulation of tubulin and other flagellar mRNAs by kinetics similar to those of controls. However, unlike controls, in which the accumulated mRNAs are rapidly degraded, these mRNAs are stabilized in cycloheximide. The stabilization by cycloheximide is specific for the flagellar mRNAs accumulated after deflagellation, since there is no change in the levels of flagellar mRNAs in nondeflagellated (uninduced) cells in the presence of cycloheximide. The kinetics of flagellar mRNA synthesis after deflagellation are shown to be the same in cycloheximide-treated and control cells by in vivo labeling and in vitro nuclear runoff experiments. These results show that protein synthesis is not required for the induced synthesis of flagellar mRNAs, and that all necessary transcriptional control factors are present in the cell before deflagellation, but that protein synthesis is required for the accelerated degradation of the accumulated flagellar mRNAs. Since cycloheximide prevents the induced synthesis and accumulation of flagellar proteins, it is possible that the product(s) of protein synthesis required for the accelerated decay of these mRNAs is a flagellar protein(s). The possibility that one or more flagellar proteins autoregulate the stability of the flagellar mRNAs is discussed.

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Biotin-mediated protein biosynthesis

Biochemical Journal ◽

10.1042/bj1400549 ◽

1974 ◽

Vol 140 (3) ◽

pp. 549-556 ◽

Cited By ~ 24

Author(s):

R. L. Boeckx ◽

K. Dakshinamurti

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Rat Liver ◽

Intestinal Mucosa ◽

Protein Biosynthesis ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Stimulation Of

The effect of administration of biotin to biotin-deficient rats on protein biosynthesis was studied. Biotin treatment resulted in stimulation by more than twofold of amino acid incorporation into protein, both in vivo and in vitro in rat liver, pancreas, intestinal mucosa and skin. Analysis of the products of amino acid incorporation into liver proteins in vivo and in vitro indicated that the synthesis of some proteins was stimulated more than twofold, but others were not stimulated at all. This indicates a specificity in the stimulation of protein synthesis mediated by biotin.

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Protein synthesis is required for rapid degradation of tubulin mRNA and other deflagellation-induced RNAs in Chlamydomonas reinhardi.

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.54 ◽

1986 ◽

Vol 6 (1) ◽

pp. 54-61 ◽

Cited By ~ 30

Author(s):

E J Baker ◽

L R Keller ◽

J A Schloss ◽

J L Rosenbaum

Keyword(s):

Protein Synthesis ◽

Transcriptional Control ◽

Protein S ◽

Protein Synthesis Inhibitors ◽

Control Factors ◽

Flagellar Proteins ◽

The Stability ◽

And Control

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Cyclin E Regulation by the Fbw7 Ubiquitin Ligase Pathway Is Required for Normal Mitophagy and Restraining Reactive Oxidative Species During Erythroid Cell Maturation In Vivo,

Blood ◽

10.1182/blood.v118.21.3393.3393 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3393-3393

Author(s):

Yanfei Xu ◽

Ka Tat Siu ◽

Amit Verma ◽

Alexander C. Minella

Keyword(s):

Ubiquitin Ligase ◽

Conflicts Of Interest ◽

Cyclin E ◽

Cell Maturation ◽

Erythroid Cell ◽

Reactive Oxidative Species ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Oxidative Species

Abstract Abstract 3393 We previously described a knock-in mouse model that permits study of the physiologic consequences of cyclin E deregulation, by selective ablation of its regulation via the SCFFbw7 ubiquitin ligase. We found that erythroid progenitor cells in our cyclin ET74A T393A knock-in mice exhibit abnormally increased proliferation, increased apoptosis, impaired maturation, and dysplastic morphologies. Most prominent among the gene expression alterations we have identified in the cyclin E knock-in erythroid cells is induction of multiple p53 target genes, consistent with p53 pathway activation. In contrast to several recently described models of ribosomal protein gene mutations, in which p53 activation appears to induce dyserythropoiesis, we determined that p53 function actually maintains partially compensated erythroid cell maturation in vivo, in the context of impaired Fbw7-mediated cyclin E degradation. We next found that dysregulated cyclin E-CDK2 activity in cyclin ET74A T393A erythroid cells is associated with increased reactive oxidative species and increased mitochondrial mass and activity. These results coincide with findings of abnormal mitochondria retention in late-stage erythroid cells and significantly down-regulated expression of BNIP3L (NIX). BNIP3L encodes a critical regulator of erythroid cell mitophagy, and the transcriptional controls maintaining its expression in maturing erythroid cells likely account for why this lineage is acutely sensitive to deregulated cyclin E activity. Finally, we show evidence that reduced expression of BNIP3L may play a role in some cases of early-stage myelodysplastic syndromes. Disclosures: No relevant conflicts of interest to declare.

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Isolation and in vitro differentiation of human erythroid precursor cells

Blood ◽

10.1182/blood.v47.5.767.bloodjournal475767 ◽

1976 ◽

Vol 47 (5) ◽

pp. 767-776

Author(s):

HC Kim ◽

PA Marks ◽

RA Rifking ◽

GM Maniatis ◽

A Bank

Keyword(s):

Precursor Cells ◽

Cell Maturation ◽

Erythroid Cell ◽

Beta Thalassemia ◽

Beta Globin ◽

Erythroid Cells ◽

Erythroid Precursor ◽

Globin Synthesis ◽

Chain Synthesis

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24–48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha- globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.

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Regulation of Erythroid Cell Maturation Is Mediated by a Foxo3-mTOR Cross Talk: Outcome for Beta-Thalassemic Erythropoiesis

Blood ◽

10.1182/blood.v118.21.176.176 ◽

2011 ◽

Vol 118 (21) ◽

pp. 176-176

Author(s):

Xin Zhang ◽

Valentina D'Escamard ◽

Pauline Rimmele ◽

Saghi Ghaffari

Keyword(s):

Oxidative Stress ◽

Signaling Pathway ◽

Mtor Signaling ◽

Cell Maturation ◽

Erythroid Cell ◽

Beta Thalassemia ◽

Erythroid Cells ◽

Mtor Signaling Pathway ◽

Erythroid Precursors

Abstract Abstract 176 Differentiation of erythroid progenitors to mature red blood cells requires erythropoietin receptor (EpoR) signaling. Stimulation of EpoR results in Jak2-mediated activation of mainly AKT, ERK/MAPK and STAT5 signaling pathways. Although alteration of these pathways is involved with the pathophysiology of major erythroid disorders such as beta-thalassemia mechanisms by which these signals impact transcriptional programs of erythroid cell maturation are largely unknown. We have shown previously that AKT signaling is required for Epo-mediated erythroid cell maturation and identified Foxo3 transcription factor, that is negatively regulated by AKT, as a critical regulator of erythroid cell cycle, maturation and lifespan mostly through the control of oxidative stress (Marinkovic et al., JCI, 2007). In addition to Foxo3, AKT regulates several proteins including the mammalian target of rapamycin (mTOR). Here we asked how Foxo3 regulation of oxidative stress impacts erythroid cell maturation. We found that AKT/mTOR signaling pathway is constitutively activated, possibly as part of a feedback loop, in primary Foxo3−/− erythroid precursors. In addition, Epo stimulation of primary Foxo3−/− erythroid precursors led to hyperphosphorylation of Jak2, AKT, mTOR and its target p70S6 Kinase (S6K) as compared to control cells. Since Foxo3 controls levels of reactive oxygen species (ROS) in erythroid cells, and ROS are known to modify signaling proteins, we asked whether ROS are involved in the hyperactivation of AKT/mTOR signaling pathway in Foxo3−/− erythroid precursors. Combined in vivo and in vitro treatment of Foxo3−/− erythroid precursors with ROS scavenger N-Acetyl-Cysteine (NAC) reduced significantly the hyper-phosphorylation of AKT, mTOR and S6K in response to Epo. These results strongly suggest that ROS mediate the hyperactivation of AKT/mTOR signaling pathway in Foxo3−/− erythroid precursors. Next we addressed whether the imbalanced production versus maturation of Foxo3−/− erythroid precursors (Marinkovic et al., JCI, 2007) is due to the constitutive activation of AKT/mTOR signaling. This was indeed the case since in vivo treatment of Foxo3−/− mice for three weeks with the mTOR inhibitor rapamycin shifted the balance from immature towards mature erythroid cells. Interestingly while rapamycin treatment decreased cycling of Foxo3−/− erythroid progenitors as anticipated, it resulted in highly increased proliferation of Foxo3−/− mature erythroblasts as analyzed by in vivo BrdU assay. Importantly, the described Foxo3−/− erythroid phenotype was maintained on two distinct genetic backgrounds (C57BL/6 and BALB/c) in mice. These results strongly suggest that the oxidative stress-induced activation of mTOR signaling pathway mediates the imbalanced production of mature erythroid cells in Foxo3−/− mice. Given that both oxidative stress and delayed erythroid cell differentiation as seen in Foxo3−/−erythroid precursors, contribute significantly to beta-thalassemia, we asked whether the mTOR signaling is involved in the pathogenesis of this disease. Rapamycin treatment improved erythroid cell maturation in the bone marrow as analyzed by cell size, CD44, TER 119 and CD71 surface markers, and resulted in significant increase in total peripheral blood red cells and hemoglobin (1 to 1.5 g/dl increase), significant reduction in reticulocyte production as well as decrease in the spleen size of beta-thalassemic intermedia (th3/+) mice similar to what was seen in Foxo3−/− mice. Collectively these results indicate an important function for the Foxo3-mTOR cross talk in the regulation of erythroid cell maturation and suggest that rapamycin may be considered for treatment of beta-thalassemia. Disclosures: No relevant conflicts of interest to declare.

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Heme-Regulated Inhibitor (HRI) Depletion Cooperates with Pharmacologic Inducers to Strongly Elevate Fetal Hemoglobin and Reduce Sickle Cell Formation

Blood ◽

10.1182/blood-2019-125664 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3557-3557

Author(s):

Scott A. Peslak ◽

Xianjiang Lan ◽

Eugene Khandros ◽

Peng Huang ◽

Jennifer A. Yano ◽

...

Keyword(s):

Sickle Cell ◽

Research Funding ◽

Fetal Hemoglobin ◽

Cell Maturation ◽

Erythroid Cell ◽

Dose Titration ◽

Cell Phenotype ◽

Erythroid Cells ◽

Hbf Induction

Increasing fetal hemoglobin (HbF) provides significant clinical benefit for patients with sickle cell disease (SCD) and is a critical goal that is being pursued with a variety of pharmacologic strategies. Hydroxyurea (HU) is currently the only FDA-approved drug aimed at raising HbF but is limited in its efficacy. Recent work in our laboratory identified heme-regulated inhibitor (HRI), an erythroid-specific protein kinase, as a novel HbF regulator (Grevet et al., Science 2018). Depleting HRI in primary human erythroid cells significantly raised HbF levels; however, maximal HbF induction by HRI knockdown required at least 80-85% HRI depletion. It is currently unknown whether this degree of HRI inhibition can be achieved pharmacologically. Besides HU, several compounds have been recently identified as in vitro HbF inducers, including pomalidomide, a third-generation imide, and UNC0638, an EHMT 1/2 histone methyltransferase inhibitor. We therefore set out to test whether combining HRI depletion with pharmacologic HbF inducers would cooperatively increase HbF levels and diminish potential adverse effects on erythroid cell maturation and viability. We performed three-stage in vitro culture of human CD34+ cells treated with select pharmacologic inducers of HbF, including HU, pomalidomide, or UNC0638, in combination with shRNA knockdown of HRI. HbF levels were assessed by RT-qPCR, Western blot, flow cytometry, and HPLC. We found that treatment with HU displayed comparatively little activity and failed to increase effects of HRI knockdown. However, treatment with UNC0638 combined with HRI depletion showed greater than additive effects on HbF with levels reaching 25-30% HbF, while HRI depletion combined with pomalidomide treatment showed the highest levels of cooperativity, reaching 30-40% HbF. Combination of HRI depletion and HbF pharmacologic inducers showed minimal effects on the erythroid transcriptome by RNA-Seq and did not significantly impair erythroid maturation. Intriguingly, dose-titration experiments indicated that HRI knockdown sensitizes erythroid cells to low doses of pomalidomide, maintaining HbF levels of greater than 40% in HRI-depleted samples despite a ten-fold decrease in pomalidomide concentration. Diminished expression of the HbF repressor BCL11A accounted in large part for HbF induction in HRI knockdown samples, particularly when combined with pomalidomide treatment in which BCL11A depletion exceeded 90%, while other HbF repressors such as LRF were unchanged. We will present RNASeq analyses aimed at elucidating mechanisms of HRI cooperativity. Finally, we found that HRI depletion in SCD patient-derived cells showed significant cooperativity with pomalidomide and UNC0638, achieving 45-50% HbF for UNC0638-treated and 50-60% HbF for pomalidomide-treated drug combinations. Importantly, combination of HRI knockdown and HbF pharmacologic treatments markedly reduced in vitro sickling as measured by low-oxygen sickling assays, suggesting significant amelioration of the sickle cell phenotype. In sum, we find that dual targeting of HbF induction via HRI inhibition and pharmacologic inducers results in successful cooperative upregulation of HbF levels in both normal and SCD primary human cells without impairing red cell maturation. Furthermore, our data suggest that dose titration of HbF inducers combined with HRI depletion could maximize HbF induction while potentially reducing off-target effects. Moreover, HRI is an attractive target for HbF induction as it is expressed in an erythroid-specific manner. While no specific HRI inhibitors are currently available, our work suggests that future small molecule inhibitors of HRI may be combined with other pharmacotherapies to achieve significant, clinically meaningful HbF induction for the treatment of SCD and other hemoglobinopathies. Disclosures Abdulmalik: The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent. Blobel:Bioverativ: Research Funding; Pfizer: Research Funding.

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BCL-XL mRNA Is Induced in Erythroid Progenitors In Vivo in a Mouse Model of Erythropoietic Stress.

Blood ◽

10.1182/blood.v108.11.1129.1129 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1129-1129

Author(s):

Ying Liu ◽

Merav Socolovsky

Keyword(s):

Flow Cytometry ◽

Stress Response ◽

Mouse Model ◽

Time Course ◽

Erythroid Cell ◽

Post Injection ◽

Erythroid Cells ◽

Stress Erythropoiesis

Abstract The rate of red blood cell production increases up to ten-fold during stress erythropoiesis. We have recently identified stress-responsive CD71highTer119positive early erythroblast subsets in freshly-isolated mouse hematopoietic tissue by flow cytometry. Both the absolute number and relative frequency of these early erythroblast subsets increase dramatically during stress. We have shown that this erythroblast expansion is associated with enhanced erythroblast viability, which is at least in part due to down-regulation of the death-receptor Fas, and its ligand, FasL from early erythroblasts by erythropoietin-receptor (EpoR) signaling (Liu et al., Blood 2006). The anti-apoptotic protein bcl-xL is induced in differentiating erythroid cells in vitro by EpoR and Stat5 signaling (Socolovsky et al., Cell 1999). Bcl-xL is essential for erythroid cell viability and is required for the maintenance of the normal basal hematocrit (Motoyama et al., Science 1995). However, it is unclear whether bcl-xL plays a role in enhancing erythroblast viability during the stress response. Serum factors other than Epo may modulate erythroid bcl-xL levels (Dolznig et al., Oncogene 2006), complicating the interpretation of bcl-xL measurements in cultured erythroid cells in vitro. Therefore, we examined the potential role of bcl-xL in stress erythropoiesis by measuring bcl-xL mRNA directly in CD71highTer119positive early erythroblasts in vivo in a mouse model of stress. We mimicked the effect of acute erythropoietic stress by injecting adult Balb/C mice with a single dose of Epo (50 mg/kg subcutaneously). Control mice were injected with an equal volume of saline. Spleen cells were harvested at 3, 16, 24, 48 and 72 hours post injection, and CD71highTer119positive early erythroblasts were immediately sorted by flow-cytometry. RNA was extracted from these freshly sorted cells and used in quantitative real-time PCR to measure bcl-xL mRNA expression. We normalized the level of bcl-xL mRNA in each sample by expressing it relative to beta-actin mRNA. At least 3 independent experiments were conducted for each time point. In parallel, we measured serum Epo concentration following Epo injection by ELISA. This showed that Epo increased approximately 100 fold by 40 minutes post-injection, reaching a peak by 6 hours and returning to basline levels by 48 hours. We found that bcl-xL mRNA began to increase in spleen early erythroblasts by 3 hours following Epo injection. By 16 hours, bcl-xL mRNA in Epo-injected mice was three-fold higher than in mice injected with saline. Bcl-xL mRNA continued to be elevated, by 2.5 fold, at 24 hours, but declined back to baseline levels by 48 hours. The time course of the increase in splenic early erythroblast bcl-xL mRNA therefore closely parallels the time course of serum Epo. The induction of early erythroblat bcl-xL mRNA suggests it is likely to contribute to the viability of stress-responsive CD71highTer119positive early erythroblasts, and therefore to the increased erythropoietic rate during the stress response.

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Isolation and in vitro differentiation of human erythroid precursor cells

Blood ◽

10.1182/blood.v47.5.767.767 ◽

1976 ◽

Vol 47 (5) ◽

pp. 767-776 ◽

Cited By ~ 4

Author(s):

HC Kim ◽

PA Marks ◽

RA Rifking ◽

GM Maniatis ◽

A Bank

Keyword(s):

Precursor Cells ◽

Cell Maturation ◽

Erythroid Cell ◽

Beta Thalassemia ◽

Beta Globin ◽

Erythroid Cells ◽

Erythroid Precursor ◽

Globin Synthesis ◽

Chain Synthesis

Abstract There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24–48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha- globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.

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Characteristics of in Vitro Differentiated Erythrocytes Derived from Human Bmi-1 Extensively Expanded Erythroblasts (E3)

Blood ◽

10.1182/blood-2020-142726 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-30

Author(s):

Moira M. Lancelot ◽

Natia Saakadze ◽

Jiusheng Deng ◽

Sean R. Stowell ◽

Ryan Jajosky ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Lentiviral Vector ◽

Culture Conditions ◽

Erythroid Cell ◽

Differentiation Process ◽

Erythroid Cells ◽

Publicly Traded

Background and significance: Blood transfusion is an important and sometimes life-saving therapeutic intervention that may not always be available due to donor shortages, alloimmunization, and rare blood groups. Red blood cells (RBCs) produced in vitro may provide an inexhaustible alternative or complimentary source of blood products for those patients without compatible donors, while eliminating the risk of infection and transfusion reactions. Bmi-1 overexpression has been shown to increase the self-renewal potential of mouse erythroblasts. We describe studies employing Bmi-1 overexpression in human erythroblasts to produce cultures of extensively expanded erythroblasts (E3) that can be differentiated into mature erythrocytes, and used for the study and optimization of ex-vivo methods of erythropoiesis with the ultimate goal of mass production of engineered blood red blood cells for transfusion purposes. Methods: Human erythroblasts, derived from peripheral blood of human donors, were transduced with a lentiviral vector expressing Bmi-1 to produce cultures of E3 cells. Following the transfer of proliferating E3 cells to differentiation culture conditions, we characterized changes in cell morphology and expression of erythroid cell surface markers CD49, CD71 and CD235a at various time points. Additionally, we tracked cells numbers, cell viability, and enucleation. At the end of the differentiation process we also analyzed cells for hemoglobinization and blood group expression and compared our results to the original donor cells. Culture conditions were optimized to increase the yield of viable and enucleated cells at the end of the differentiation process. Findings: In vitro maturation and differentiation of Bmi-1-E3 cells is characterized by progressive enucleation (up to 50% of total erythroid cells in some experiments), as well as down-regulation of transferrin receptor (CD71) and α4 integrin (CD49d). As maturation progressed, cells also underwent additional changes including decrease in size, nuclear condensation, and accumulation of hemoglobin in the cytoplasm. We were able to optimize conditions to demonstrate that cells were terminally maturing over time in the culture system. Conclusion(s): The data shows that Bmi-1-E3 cells can be cultured in vitro to produce mature erythroid cells with characteristics desirable for transfusion for those patients who may not otherwise have a suitable source for transfusion. Further work is required in order to increase enucleation and evaluate the survival and functionality of these cells in vivo, as once completely enucleated, cultured red cells do not pose concerns over genotoxicity and should be of adequate quality to be used for transfusion purposes. Simultaneously, our group is developing a mouse model to test the in-vivo performance of these cultured red blood cells. Disclosures Jajosky: Biconcavity Inc.: Other: CEO and partial owner; BioMarin Pharmaceuticals: Current equity holder in publicly-traded company; Magenta Therapeutics: Current equity holder in publicly-traded company.

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