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1.  Iron dose-dependent differentiation and enucleation of human erythroblasts in serum-free medium 
Improvements in ex vivo generation of enucleated red blood cells are being sought for erythroid biology research, toward the ultimate goal of erythrocyte engineering for clinical use. Based upon the high levels of iron-saturated transferrin in plasma serum, it was hypothesized that terminal differentiation in serum-free media may be highly dependent on the concentration of iron. Here adult human CD34+ cells were cultured in a serum-free medium containing dosed levels of iron-saturated transferrin (holo-Tf, 0.1–1.0 mg/ml). Iron in the culture medium was reduced, but not depleted, with erythroblast differentiation into haemoglobinized cells. At the lowest holo-Tf dose (0.1 mg/ml), terminal differentiation was significantly reduced and the majority of the cells underwent apoptotic death. Cell survival, differentiation and enucleation were enhanced as the holo-Tf dose increased. These data suggest that adequate holo-Tf dosing is critical for terminal differentiation and enucleation of human erythroblasts generated ex vivo in serum-free culture conditions. Published 2013. This article is a US Government work and is in the public domain in the USA.
doi:10.1002/term.1743
PMCID: PMC3883763  PMID: 23606586
erythropoiesis; serum-free media; holotransferrin; haemoglobin; enucleation; iron
2.  LIN28A Expression Reduces Sickling of Cultured Human Erythrocytes 
PLoS ONE  2014;9(9):e106924.
Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with sickle cell disease (SCD) and the beta-thalassemias. It was recently reported that increased expression of LIN28 proteins or decreased expression of its target let-7 miRNAs enhances HbF levels in cultured primary human erythroblasts from adult healthy donors. Here LIN28A effects were studied further using erythrocytes cultured from peripheral blood progenitor cells of pediatric subjects with SCD. Transgenic expression of LIN28A was accomplished by lentiviral transduction in CD34(+) sickle cells cultivated ex vivo in serum-free medium. LIN28A over-expression (LIN28A-OE) increased HbF, reduced beta (sickle)-globin, and strongly suppressed all members of the let-7 family of miRNAs. LIN28A-OE did not affect erythroblast differentiation or prevent enucleation, but it significantly reduced or ameliorated the sickling morphologies of the enucleated erythrocytes.
doi:10.1371/journal.pone.0106924
PMCID: PMC4154803  PMID: 25188417
3.  Increased Reticulocytosis during Infancy Is Associated with Increased Hospitalizations in Sickle Cell Anemia Patients during the First Three Years of Life 
PLoS ONE  2013;8(8):e70794.
Objective
Among older children with sickle cell anemia, leukocyte counts, hemoglobin, and reticulocytosis have previously been suggested as disease severity markers. Here we explored whether these blood parameters may be useful to predict early childhood disease severity when tested in early infancy, defined as postnatal ages 60–180 days.
Study Design
Data from fifty-nine subjects who were followed at Children’s National Medical Center’s Sickle Cell Program for at least three years was retrospectively analyzed. Comparisons were made between white blood cell counts, hemoglobin and reticulocyte levels measured at ages 60–180 days and the clinical course of sickle cell anemia during infancy and childhood.
Results
A majority of subjects had demonstrable anemia with increased reticulocytosis. Only increased absolute reticulocyte levels during early infancy were associated with a significant increase in hospitalization during the first three years of life. Higher absolute reticulocyte counts were also associated with a markedly shorter time to first hospitalizations and a four-fold higher cumulative frequency of clinical manifestations over the first three years of life. No significant increase in white blood cell counts was identified among the infant subjects.
Conclusions
These data suggest that during early infancy, increased reticulocytosis among asymptomatic SCA subjects is associated with increased severity of disease in childhood.
doi:10.1371/journal.pone.0070794
PMCID: PMC3737358  PMID: 23951011
4.  A Synthetic Model of Human Beta-Thalassemia Erythropoiesis Using CD34+ Cells from Healthy Adult Donors 
PLoS ONE  2013;8(7):e68307.
Based upon the lack of clinical samples available for research in many laboratories worldwide, a significant gap exists between basic and clinical studies of beta-thalassemia major. To bridge this gap, we developed an artificially engineered model for human beta thalassemia by knocking down beta-globin gene and protein expression in cultured CD34+ cells obtained from healthy adults. Lentiviral-mediated transduction of beta-globin shRNA (beta-KD) caused imbalanced globin chain production. Beta-globin mRNA was reduced by 90% compared to controls, while alpha-globin mRNA levels were maintained. HPLC analyses revealed a 96% reduction in HbA with only a minor increase in HbF. During the terminal phases of differentiation (culture days 14–21), beta-KD cells demonstrated increased levels of insoluble alpha-globin, as well as activated caspase-3. The majority of the beta-KD cells underwent apoptosis around the polychromatophilic stage of maturation. GDF15, a marker of ineffective erythropoiesis in humans with thalassemia, was significantly increased in the culture supernatants from the beta-KD cells. Knockdown of beta-globin expression in cultured primary human erythroblasts provides a robust ex vivo model for beta-thalassemia.
doi:10.1371/journal.pone.0068307
PMCID: PMC3704632  PMID: 23861885
5.  Expression of growth differentiation factor 15 is not elevated in individuals with iron deficiency secondary to volunteer blood donation 
Transfusion  2010;50(7):1532-1535.
BACKGROUND
Low serum hepcidin levels provide a physiologic response to iron demand in patients with iron deficiency (ID). Based on a discovery of suppressed hepcidin expression by a cytokine named growth differentiation factor 15 (GDF15), it was hypothesized that GDF15 may suppress hepcidin expression in humans with ID due to blood loss.
STUDY DESIGN AND METHODS
To test this hypothesis, GDF15 and hepcidin levels were measured in peripheral blood from subjects with iron-deficient erythropoiesis before and after iron supplementation.
RESULTS
Iron variables and hepcidin levels were significantly suppressed in iron-deficient blood donors compared to healthy volunteers. However, ID was not associated with elevated serum levels of GDF15. Instead, iron-deficient subjects’ GDF15 levels were slightly lower than those measured in the control group of subjects (307 ± 90 and 386 ± 104 pg/mL, respectively). Additionally, GDF15 levels were not significantly altered by iron repletion.
CONCLUSIONS
ID due to blood loss is not associated with a significant change in serum levels of GDF15.
doi:10.1111/j.1537-2995.2010.02601.x
PMCID: PMC3282986  PMID: 20210929
6.  HEPCIDIN, ANAEMIA, AND PROSTATE CANCER 
Bju International  2011;107(4):678-679.
doi:10.1111/j.1464-410X.2011.10108.x
PMCID: PMC3277454  PMID: 21276178
7.  Let-7 microRNAs are developmentally regulated in circulating human erythroid cells 
Background
MicroRNAs are ~22nt-long small non-coding RNAs that negatively regulate protein expression through mRNA degradation or translational repression in eukaryotic cells. Based upon their importance in regulating development and terminal differentiation in model systems, erythrocyte microRNA profiles were examined at birth and in adults to determine if changes in their abundance coincide with the developmental phenomenon of hemoglobin switching.
Methods
Expression profiling of microRNA was performed using total RNA from four adult peripheral blood samples compared to four cord blood samples after depletion of plasma, platelets, and nucleated cells. Labeled RNAs were hybridized to custom spotted arrays containing 474 human microRNA species (miRBase release 9.1). Total RNA from Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines provided a hybridization reference for all samples to generate microRNA abundance profile for each sample.
Results
Among 206 detected miRNAs, 79% of the microRNAs were present at equivalent levels in both cord and adult cells. By comparison, 37 microRNAs were up-regulated and 4 microRNAs were down-regulated in adult erythroid cells (fold change > 2; p < 0.01). Among the up-regulated subset, the let-7 miRNA family consistently demonstrated increased abundance in the adult samples by array-based analyses that were confirmed by quantitative PCR (4.5 to 18.4 fold increases in 6 of 8 let-7 miRNA). Profiling studies of messenger RNA (mRNA) in these cells additionally demonstrated down-regulation of ten let-7 target genes in the adult cells.
Conclusion
These data suggest that a consistent pattern of up-regulation among let-7 miRNA in circulating erythroid cells occurs in association with hemoglobin switching during the fetal-to-adult developmental transition in humans.
doi:10.1186/1479-5876-7-98
PMCID: PMC2792219  PMID: 19939273
8.  Inhibition of erythroblast growth and fetal hemoglobin production by ribofuranose-substituted adenosine derivatives 
Biochimica et biophysica acta  2008;1782(9):504-510.
In vivo, inhibition of fetal hemoglobin (HbF) expression in humans around the time of birth causes the clinical manifestation of sickle cell and beta-thalassemia syndromes. Inhibition of HbF among cultured cells was recently described by the adenosine derivative molecule named SQ22536. Here, a primary cell culture model was utilized to further explore the inhibition of HbF by adenosine derivative molecules. SQ22536 demonstrated down-regulation of growth and HbF expression among erythroblasts cultured from fetal and adult human blood. The effects upon HbF were noted in a majority of cells, and quantitative PCR analysis demonstrated a transcriptional mechanism. Screening assays demonstrated two additional molecules named 5′-deoxy adenosine and 2′,3′-dideoxy adenosine had effects on HbF comparable to SQ22536. Other adenosine-derivative molecules, adenosine receptor binding ligands, and cAMP-signaling regulators failed to inhibit HbF in matched cultures. These results suggest structurally-related ribofuranose-substituted adenosine analogues act through an unknown mechanism to inhibit HbF expression in fetal and adult human erythroblasts.
doi:10.1016/j.bbadis.2008.05.004
PMCID: PMC2613185  PMID: 18586086
Human erythropoiesis; cytokines; HbF inhibition; adenosine derivatives; SQ22536; hemoglobinopathies
9.  Hembase: browser and genome portal for hematology and erythroid biology 
Nucleic Acids Research  2004;32(Database issue):D572-D574.
Hembase (http://hembase.niddk.nih.gov) is an integrated browser and genome portal designed for web-based examination of the human erythroid transcriptome. To date, Hembase contains 15 752 entries from erythroblast Expressed Sequenced Tags (ESTs) and 380 referenced genes relevant for erythropoiesis. The database is organized to provide a cytogenetic band position, a unique name as well as a concise annotation for each entry. Search queries may be performed by name, keyword or cytogenetic location. Search results are linked to primary sequence data and three major human genome browsers for access to information considered current at the time of each search. Hembase provides interested scientists and clinical hematologists with a genome-based approach toward the study of erythroid biology.
doi:10.1093/nar/gkh129
PMCID: PMC308863  PMID: 14681483

Results 1-9 (9)