Ineffective Erythropoiesis and Production of Normoblasts with a Beta Thalassemia Major Phenotype Using CD34+ Cells From Healthy Donors

Abstract Abstract 1085 Reversal of anemia is the major target of thalassemia research, but studies of the molecular and cellular basis of the ineffective erythropoiesis of thalassemia are limited by access to donor progenitor cells. Here we demonstrate that thalassemic erythropoiesis may be recapitulated ex vivo by reducing the expression of hemoglobin in cultured CD34+ cells. Using lentiviral transduction of progenitor cells obtained from three healthy adult human donors, shRNA molecules were screened for their ability to reduce beta-globin gene and protein expression over 21 days in culture. Cells transduced with a scrambled vector served as donor-matched controls. Among the screened shRNA, one named HBB caused a consistent and significant reduction in beta-globin mRNA and protein. Beta-globin mRNA was reduced to levels <10% (p<0.001) compared to that of the controls (day14/21), while maintaining expression of gamma- and alpha-globin mRNA. HPLC was performed on an equivalent number of cells sampled on culture day 21 for hemoglobin type (HbA vs. HbF) and quantitation (area under each HPLC peak). The HbA peak was reduced by 96%, and there was a minor increase in the HbF peak (1.6 fold) after HBB transduction. Based upon these quantitative changes in hemoglobin, HbF represented 49.3±9.3% in the HBB transduced population compared with 2.9±0.7% (p<0.01) in controls. On culture day 14, there was no significant difference in glycophorin A (CD235), transferrin receptor (CD71), or cellular morphology despite the reduction in beta-globin mRNA. However, impaired terminal differentiation was detected by retainment of surface CD71 and a lack of enucleation during the third week of culture. Cell counts were lower in HBB transduced cells during the final stages of erythroid differentiation with a 61% (p=0.03) reduction in total cell counts by day 21 when compared to controls. Annexin V assay on day 21 also demonstrated increased phosphatidylserine expression in the HBB transduced cells [HBB=55.7±14.4% vs. Control=25.0±3.0%] in association with the decreased terminal differentiation. GDF15 quantitation demonstrated a significant (p=0.006) increase in the culture supernatants of HBB transduced cells. Sorted cytospin preparations revealed a distinct population of mature normoblasts containing a highly condensed nucleus surrounded by a thin ring of hypochromic cytoplasm. Reduction of erythroblast beta-globin gene and protein expression to levels associated with beta thalassemia major in humans causes ineffective erythropoiesis ex vivo by reducing cell production, increasing surface expression of phosphatidylserine, and impairing enucleation during terminal maturation. Efforts are now underway to use the culture system to explore mechanisms whereby reduced hemoglobin synthesis causes normoblast defects, and for screening of chemical and genetic rescue therapies for the thalassemic erythroid phenotype. Disclosures: No relevant conflicts of interest to declare.

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IGF2BP1 Reverses Hemoglobin Switching in Adult Erythroblasts

Blood ◽

10.1182/blood.v126.23.639.639 ◽

2015 ◽

Vol 126 (23) ◽

pp. 639-639

Author(s):

Laxminath Tumburu ◽

Colleen Byrnes ◽

Y. Terry Lee ◽

Jaira F. de Vasconcellos ◽

Antoinette Rabel ◽

...

Keyword(s):

Gene Expression ◽

Adult Stage ◽

Globin Gene ◽

Expression Patterns ◽

Beta Globin ◽

Globin Mrna ◽

Beta Globin Gene ◽

Empty Vector ◽

Cd34 Cells ◽

Gene And Protein Expression

Abstract During human ontogeny, high-level transcription within the beta-globin gene cluster switches sequentially from embryonic-to-fetal-to-adult genes. Beta-thalassemias and sickle-cell disease are manifested by reduced or mutated expression of the adult-stage, beta-globin gene. Research is aimed toward the eventual therapeutic goal of safely preventing or reversing the fetal-to-adult hemoglobin switch among these patient populations. To identify genes that may be involved in regulation of the fetal-to-adult erythroid switch, purified CD34(+) cells from six umbilical cord (fetal) and six adult peripheral blood samples were cultured in serum-free medium, and gene expression libraries were prepared and sequenced from CD71(+), CD235a(+) erythroblast mRNA. In total, 546 million paired-end reads with a length of 101bp were generated for a comparison of cord and adult erythroblast transcriptomes. Reads were aligned to the human reference genome (hg19), and differential gene expression was identified [false discovery rate ≤ 0.05, fold change ≥ 1.5, and reads per kilobase per million mapped reads (RPKM) ≥ 0.5]. A total of 145 genes were differentially expressed according to these criteria, with four of the top five encoding targets of the let-7 family of microRNAs. The topmost gene was insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which is normally involved in transcriptome regulation and developmental timing. IGF2BP1 expression was 770-fold increased in the fetal erythroblasts (RPKM > 3.0) compared with low background levels in adult erythroblasts (RPKM < 0.01). IGF2BP1 protein is present in fetal tissues including fetal liver; however, it is not detected in adult human bone marrow. A potential role for adult-stage IGF2BP1 over-expression (IGF2BP1-OE) in the regulation of globin genes and proteins was explored using lentiviral vectors designed for let-7 resistant, erythroid-specific expression of IGF2BP1 protein. IGF2BP1-OE transduced CD34(+) cells expressed the transgenic protein and maintained their ability to differentiate, accumulate hemoglobin, and enucleate ex vivo in the presence of erythropoietin. Globin mRNA and protein levels were investigated. While alpha-globin mRNA remained unchanged, gamma-globin mRNA became predominant [90% of (gamma + beta) mRNA] in IGF2BP1-OE samples [Control (empty vector) = 3.2E+06 ± 8.2E+05 copies/ng; IGF2BP1-OE = 2.0E+07 ± 5.9E+06 copies/ng; p < 0.05], and beta-globin mRNA decreased to minor levels [Control (empty vector) = 2.2E+07 ± 4.0E+06 copies/ng; IGF2BP1-OE = 2.2E+06 ± 6.2E+05 copies/ng; p < 0.05]. IGF2BP1-OE caused a pan-cellular HbF distribution by flow cytometry. Cellular fetal hemoglobin percentages [HbF/(HbF + HbA)] were measured as 5.3 ± 0.4% in donor matched control cells versus 80.3 ± 3.7% in IGF2BP1-OE cells (p < 0.05). HPLC tracings revealed that the minor HbA2 peak, composed of alpha and delta globin chains, was reduced or absent in IGF2BP1-OE. Also, IGF2BP1-OE suppressed the expression of related genes including the transcription factor BCL11A. These data demonstrate that erythroblast IGF2BP1 is silenced in humans during fetal-to-adult ontogeny, and that IGF2BP1 in adult erythroblasts reverses the developmentally related switch in beta-like globin gene and protein expression patterns. Disclosures No relevant conflicts of interest to declare.

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Current Perspective of Beta Thalassemia and Its Treatment Strategies

10.20944/preprints201907.0277.v1 ◽

2019 ◽

Author(s):

Shaukat Ali ◽

Shumaila Mumtaz ◽

Hafiz Abdullah Shakir ◽

Hafiz Muhammad Tahir ◽

Tafail Akbar Mughal

Keyword(s):

Genetic Testing ◽

Blood Transfusion ◽

Dna Analysis ◽

Complete Blood Count ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Hematopoietic Stem ◽

Thalassemia Minor

Thalassemia is genetic blood disease cause by absence or decrease of one or more of the globin chain synthesis. Beta thalassemia is characterized by one or more mutations in beta globin gene. Absence or reduced amount the of beta globin chains cause ineffective erythropoiesis which leads to anemia. Beta thalassemia has been further divided into three main forms: Thalassemia minor/silent carrier, major and intermedia. More severe form is thalassemia major in which patients depend upon blood transfusion for survival and high level of iron occur as a consequence of consistent blood transfusion. Over loaded iron invokes the synthesis of reactive oxygen species that are toxic in redundancy and triggering the impairment to vascular, endocrine and hepatic system. Thalassemia can be diagnosed and detected through various laboratory tests such as blood smear, prenatal testing (genetic testing of amniotic fluid), DNA analysis (genetic testing) and complete blood count. Treatment of thalassemia intermedia is symptomatic but it can also be managed by splenectomy and folic supplementation. While thalassemia major can be treated by transplantation of bone marrow, regular transfusion of blood and iron chelation treatment, stimulation of fetal hemoglobin production, hematopoietic stem cell transplantation and gene therapy.

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A first case of hemoglobin Castilla [Beta 32(B14) Leu>Arg; HBB: c.98T>G] associated with [IVS-I-1 (G>A); HBB:c.92+1G>A] mutation found in a Syrian betathalassemia patient

Thalassemia Reports ◽

10.4081/thal.2020.8396 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Ahmad Shoujaa ◽

Yasser Mukhalalaty ◽

Hossam Murad ◽

Faizeh Al-Quobaili

Keyword(s):

Molecular Analysis ◽

Clinical Case ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Blood Transfusions ◽

Beta Globin ◽

First Report ◽

Beta Thalassemia Major ◽

First Case

Beta thalassemia (β-thal) is one of the most common worldwide inherited hemoglobinopathies. Proper identification and diagnosis of hemoglobin (Hb) variants provide a major challenge. In this report, we describe a 1-year-old boy, presented with the diagnosis of β-TM (beta thalassemia major), has received regular blood transfusions. The molecular analysis revealed the presence of rare Hb Castilla [Beta 32(B14) Leu>Arg; HBB: c.98T>G] variant associated with β0 [IVS-I-1 (G>A); AG^GTTGGT- >AGATTGGT beta0] (HBB:c.92+1G>A) Mutation in beta-globin (β-globin) gene. To our knowledge, this is the first report of Hb Castilla [Beta 32(B14) Leu>Arg] in ExonII of β-globin gene which were found in Syrian male proband. However, we should investigate abnormal hemoglobins in patients with beta thalassemia to determine whether they have involvement with β-thalassemia mutations in the clinical case of the patients or not.

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Hypomethylation of DNA derived from purified human erythroid cells correlates with gene activity of the beta-globin cluster

Blood ◽

10.1182/blood.v66.5.1202.1202 ◽

1985 ◽

Vol 66 (5) ◽

pp. 1202-1207 ◽

Cited By ~ 19

Author(s):

A Oppenheim ◽

Y Katzir ◽

E Fibach ◽

A Goldfarb ◽

E Rachmilewitz

Keyword(s):

Peripheral Blood ◽

Globin Gene ◽

Genetic Regulation ◽

Inverse Correlation ◽

Thalassemia Major ◽

Gene Activity ◽

Beta Thalassemia ◽

Globin Genes ◽

Beta Globin ◽

A Site

Abstract Analysis of methylation at the beta-globin gene cluster was carried out on DNA derived from nucleated RBCs (orthochromatic normoblasts) isolated from peripheral blood of patients with beta-thalassemia major or other congenital hemolytic anemia after splenectomy. A procedure to separate these normoblasts from the other nucleated cells of the peripheral blood was developed, providing us with a convenient source of DNA for investigating parameters related to human erythroid differentiation. Blood samples were obtained from six adult patients who express their gamma-globin genes at different levels. Inverse correlation between methylation and gene activity was consistently observed for five of the eight sites analyzed. A site 3′ to the beta gene was always unmethylated, two sites flanking the epsilon gene were always found to be methylated, and two sites 5′ to the two gamma genes, G gamma and A gamma, were hypomethylated in correlation with gamma gene activity of the individual patients. A site 5′ to the delta gene was unmethylated in normoblasts as well as in WBC. No apparent relation between hypomethylation and gene activity was observed for two additional sites. The results suggest that methylation at specific chromosomal locations participate in genetic regulation of the beta- like globin genes in humans.

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Molecular analysis of beta zero-thalassemia intermedia in Sardinia

Blood ◽

10.1182/blood.v74.2.823.823 ◽

1989 ◽

Vol 74 (2) ◽

pp. 823-827 ◽

Cited By ~ 42

Author(s):

R Galanello ◽

E Dessi ◽

MA Melis ◽

M Addis ◽

MA Sanna ◽

...

Keyword(s):

Nonsense Mutation ◽

Frameshift Mutation ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Thalassemia Intermedia ◽

Mild Phenotype ◽

Beta Globin Gene ◽

Alpha Thalassemia

Abstract In this study we have carried out alpha- and beta-globin gene analysis and defined the beta-globin gene polymorphisms in a group of patients with thalassemia intermedia of Sardinian descent. A group of patients (109) with thalassemia major of the same origin served as control. Characterization of the beta-thalassemia mutation showed either a frameshift mutation at codon 6 or a codon 39 nonsense mutation. We found that homozygotes for the frameshift mutation at codon 6 or compound heterozygotes for this mutation and for the codon 39 nonsense mutation develop thalassemia intermedia more frequently than thalassemia major. The frameshift mutation at codon 6 was associated with haplotype IX that contains the C-T change at position -158 5′ to the G gamma globin gene implicated in high gamma chain production and thus the mild phenotype. In patients' homozygotes for codon 39 nonsense mutation, those with thalassemia intermedia more frequently had the two- gene deletion form of alpha-thalassemia, or functional loss of the alpha 2 gene as compared with those with thalassemia major. In a few siblings with thalassemia major and intermedia, the thalassemia intermedia syndrome correlated with the presence of the -alpha/-alpha genotype. No cause for the mild phenotype was detected in the majority of patients who had not inherited either haplotype IX or alpha- thalassemia.

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In silico study on RNA structures of intronic mutations of beta-globin gene

F1000Research ◽

10.12688/f1000research.21953.3 ◽

2020 ◽

Vol 9 ◽

pp. 49

Author(s):

Nur Imaniati Sumantri ◽

Kenny Lischer ◽

Dian Rachma Wijayanti ◽

Tomy Abuzairi

Keyword(s):

In Silico ◽

Rna Structure ◽

Tertiary Structure ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Rna Structures ◽

Beta Globin Gene ◽

Mrna Structure

Background: Mutation of the beta-globin gene (HBB) interferes with primary mRNA transcription, leading to beta-thalassemia disease. The IVS1nt1 and IVS1nt5 mutations were reported as two of the most prevalent intronic mutations associated with beta-thalassemia major. These mutations may affect the mRNA structure of the human beta-globin (HBB) gene. However, the mechanism by which variation in HBB alters the mRNA structure remains unclear. The objective of this study was to unveil the secondary and tertiary conformation difference of the mutants compared to the wildtype using in silico analysis. Methods: The sequence of HBB was obtained from Ensemble database and mutated manually at nucleotides 143 (IVS1nt1G>T) and 147 (IVS1nt5G>C). The RNA secondary and tertiary structure were performed by ViennaRNA Web Services and 3dRNA v2.0, respectively. Results and Discussion: The results revealed the unique folding characteristics of each mutations for the secondary and tertiary structures. Based on the structure, unwanted folding occurred in the IVS1nt1G>T and IVS1nt5G>C mRNA structures compared to the wild-type structure. This finding was supported by the results of centroid-based analysis and RNA structure analysis, indicating that the larger loops in IVS1nt1 and IVS1nt5 result in an unstable structure. Our study found that intronic mutations affect the mRNA structure of HBB by altering its folding mechanism.

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In silico study on RNA structures of intronic mutations of beta-globin gene

F1000Research ◽

10.12688/f1000research.21953.1 ◽

2020 ◽

Vol 9 ◽

pp. 49

Author(s):

Nur Imaniati Sumantri ◽

Kenny Lischer ◽

Dian Rachma Wijayanti ◽

Tomy Abuzairi

Keyword(s):

In Silico ◽

Rna Structure ◽

Tertiary Structure ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Rna Structures ◽

Beta Globin Gene ◽

Mrna Structure

Background: Mutation of the beta-globin gene (HBB) interferes with primary mRNA transcription, leading to beta-thalassemia disease. The IVS1nt1 and IVS1nt5 mutations were reported as two of the most prevalent intronic mutations associated with beta-thalassemia major. These mutations may affect the mRNA structure of the human beta-globin (HBB) gene. However, the mechanism by which variation in HBB alters the mRNA structure remains unclear. The objective of this study was to unveil the secondary and tertiary conformation difference of the mutants compared to the wildtype using in silico analysis. Methods: The sequence of HBB was obtained from Ensemble database and mutated manually at nucleotides 143 (IVS1nt1G>T) and 147 (IVS1nt5G>C). The RNA secondary and tertiary structure were performed by ViennaRNA Web Services and RNA Composer, respectively. Results and Discussion: The results revealed the unique folding characteristics of each mutations for the secondary and tertiary structures. Based on the structure, unwanted folding occurred in the IVS1nt1G>T and IVS1nt5G>C mRNA structures compared to the wild-type structure. This finding was supported by the results of centroid-based analysis and RNA structure analysis, indicating that the larger loops in IVS1nt1 and IVS1nt5 result in an unstable structure. Our study found that intronic mutations affect the mRNA structure of HBB by altering its folding mechanism.

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Investigating Zeta Globin Gene Expression to Develop a Potential Therapy for Alpha Thalassemia Major

Blood ◽

10.1182/blood-2020-142922 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 3-4

Author(s):

Georgia L. Gregory ◽

Beeke Wienert ◽

Marisa Schwab ◽

Billie Rachael Lianoglou ◽

Roger P. Hollis ◽

...

Keyword(s):

Board Of Directors ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Wild Type ◽

Advisory Committees ◽

Alpha Thalassemia ◽

Alpha Globin ◽

Zeta Globin Gene

Introduction: Alpha globin mutations are very common worldwide, and the severity of resulting anemia depends on the number and type of mutated alleles. While the 4 gene mutation (alpha thalassemia major, ATM) was previously deemed fatal except in rare cases, emerging evidence indicates that survival to birth and good postnatal outcomes are possible with in utero transfusions. We hypothesized that the embryonic zeta globin gene, which is expressed early in gestation prior to alpha globin, may compensate for the lack of alpha globin and that induction of zeta globin after it has naturally been silenced may become a new therapy for patients with ATM. Methods: We evaluated mutations in the UCSF international registry of patients with ATM to understand factors related to patient survival with and without in utero transfusions. We then engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP-2 cells) carrying the common SEA alpha globin deletion, in which zeta globin expression is preserved (H-SEA), as well as those on which the zeta globin genes were deleted (HBZ-/-) using CRISPR-Cas9. We evaluated the expression of alpha and zeta globins using qPCR, Western blot, and flow cytometry. We generated lentiviral vectors expressing zeta globin under the control of beta-globin promoters to examine changes in both zeta and alpha globin in a dynamic way. Results: None of the registry patients who survived to birth spontaneously (n=11) had a mutation that involves a concomitant deletion in zeta globin (such as the -FIL, -THAI, or -MEDII mutation), while alpha globin mutations extending into the zeta globin gene were found in 14 of 37 (38%) patients who were diagnosed prenatally, suggesting that the presence of zeta globin may play a role in the ability to survive to birth without fetal therapy. Interestingly, we found that H-SEA clones express higher levels of zeta globin than WT cells, as illustrated by quantitative real-time PCR (Fig 1A), Western blot (Fig 1B) and flow cytometry (Fig 1C). These cells also developed beta globin dimers due to excess unpaired beta-globin chains, as demonstrated by Western blotting with and without reducing agents, indicating that they are an appropriate cell model for ATM. We next generated HUDEP-2 clones lacking zeta globin (HBZ KO) and transduced these clones with lentiviral vectors expressing high levels of zeta globin (lenti-zeta) (Fig 1D). Western blotting revealed that increasing the levels of zeta globin in these cells resulted in decreased expression of alpha globin, suggesting reciprocal control between these genes (Fig 1E). Most importantly, we saw a reduction in toxic beta-globin dimers in H-SEA cells expressing high levels of zeta-globin after transduction with lenti-zeta, suggesting that zeta globin could functionally replace the missing alpha-globin (Fig 1 F,G). To understand transcriptomic differences in H-SEA cells that may result in increased zeta globin expression, we performed bulk RNA sequencing of wild type and H-SEA clones. We confirmed that zeta expression is significantly upregulated in H-SEA compared to wild type (log2 fold change of 4.25, p=2.24E-38). Pathway analysis of differentially expressed genes is ongoing. Conclusions: Our international patient registry suggests that expression of zeta globin may play a role in the spontaneous survival to birth in a subset of patients. Zeta globin expression is increased in the setting of H-SEA cells in vitro, and restoration of zeta expression by lentivirus results in a reduction of toxic beta globin dimers in these ATM cells. Furthermore, expressing zeta globin at high levels in H-WT cells decreased alpha globin expression, suggesting a reciprocal regulation of these two genes. This concept is similar to the relationship between fetal gamma and adult beta globins which has been exploited for therapeutic editing approaches in patients with beta-thalassemia. At this point, the natural repressor of zeta globin is not yet known, but our data suggests that a strategy of upregulating zeta globin could potentially be developed to mimic the ongoing trials of using the BCL11A repressor to induce gamma globin in patients with beta thalassemia and sickle cell disease. Disclosures Wienert: Integral Medicines: Current Employment. Kohn:Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. MacKenzie:Acrigen: Membership on an entity's Board of Directors or advisory committees; Ultragenyx: Research Funding.

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In silico study on RNA structures of intronic mutations of beta-globin gene

F1000Research ◽

10.12688/f1000research.21953.2 ◽

2020 ◽

Vol 9 ◽

pp. 49

Author(s):

Nur Imaniati Sumantri ◽

Kenny Lischer ◽

Dian Rachma Wijayanti ◽

Tomy Abuzairi

Keyword(s):

In Silico ◽

Rna Structure ◽

Tertiary Structure ◽

Globin Gene ◽

Thalassemia Major ◽

Beta Thalassemia ◽

Beta Globin ◽

Rna Structures ◽

Beta Globin Gene ◽

Mrna Structure

Background: Mutation of the beta-globin gene (HBB) interferes with primary mRNA transcription, leading to beta-thalassemia disease. The IVS1nt1 and IVS1nt5 mutations were reported as two of the most prevalent intronic mutations associated with beta-thalassemia major. These mutations may affect the mRNA structure of the human beta-globin (HBB) gene. However, the mechanism by which variation in HBB alters the mRNA structure remains unclear. The objective of this study was to unveil the secondary and tertiary conformation difference of the mutants compared to the wildtype using in silico analysis. Methods: The sequence of HBB was obtained from Ensemble database and mutated manually at nucleotides 143 (IVS1nt1G>T) and 147 (IVS1nt5G>C). The RNA secondary and tertiary structure were performed by ViennaRNA Web Services and RNA Composer, respectively. Results and Discussion: The results revealed the unique folding characteristics of each mutations for the secondary and tertiary structures. Based on the structure, unwanted folding occurred in the IVS1nt1G>T and IVS1nt5G>C mRNA structures compared to the wild-type structure. This finding was supported by the results of centroid-based analysis and RNA structure analysis, indicating that the larger loops in IVS1nt1 and IVS1nt5 result in an unstable structure. Our study found that intronic mutations affect the mRNA structure of HBB by altering its folding mechanism.

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Mitosis Is Not Required for Coordinated Changes in Iron Metabolism and High-Level Globin Gene Transcription in Erythropoietin-Responsive CD34+ Cells.

Blood ◽

10.1182/blood.v110.11.1776.1776 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1776-1776

Author(s):

Ajoy V. Bhupatiraju ◽

Nicole M. Josleyn ◽

Y. Terry Lee ◽

Jeffery L. Miller

Keyword(s):

Cell Division ◽

Globin Gene ◽

Beta Globin ◽

Globin Mrna ◽

Gamma Globin ◽

Cell Divisions ◽

Adult Human ◽

Human Cd34 ◽

Cd34 Cells ◽

Very High

Abstract Within the erythroid lineage, erythropoietin (EPO) responsiveness is manifested by cell division and growth as well as specific changes in heme and globin production that ultimately result in the production of erythrocytes. However, the nascent relationship between EPO-associated mitosis and globin gene regulation has not been fully defined. In this study, cultured adult human CD34+ cells from peripheral blood were used to investigate early cellular responses to erythropoietin in the context of mitosis. Matched cultures were performed in replicate using human cells from at least two healthy adults. To detect mitosis, one million cells were labeled with 2uM carboxyfluorescein diacetate succinamidyl ester (CFSE). The CFSE-labeled cells were then cultured in the presence [EPO(+)] or absence [EPO(−)] of 4U/mL EPO, and analyzed using flow cytometry. No cell divisions were detected in either condition during the first 24 hours in culture, and multiple cell divisions were noted on subsequent culture days only in the EPO(+) cultures. Remarkably, dual-staining with CFSE and CD71 revealed a small (<1%) population of the undivided cells at 24 hours with very high surface levels of transferrin receptor [CD71(++++)] exclusively in the EPO(+) cultures. Further analysis of those rare EPO-responsive, pre-mitotic cells revealed DNA synthesis and entry into the cell cycle in 62.5±4.7% compared with 1.5±1.8% among the cells with lower CD71 expression. None expressed glycophorin A. Based upon their distinct phenotype, single cells were sorted into 96-well plates, with sorting confirmation by quantitative RT-PCR of GAPDH mRNA (20 copies/cell detection limit). Next, gamma- and beta-globin transcripts were amplified for comparison (4 separate experiments). Among the pre-mitotic, CD71(++++) population, 20 of 182 total sorted cells (11%) lacked detectable levels of gamma- and beta-globin mRNA. Only 1 of 182 cells (0.5%) expressed gamma-globin mRNA and no detectable beta globin mRNA. 138 of 182 cells (76%) expressed only beta-globin mRNA, and 23 of 182 cells (13%) expressed both globin mRNAs. The median and mean levels of gamma-globin mRNA among the 24 gamma(+) cells were 92 and 245±1015 copies/cell respectively. In contrast, the median and mean levels of beta-globin mRNA among the 161 beta(+) cells were 1624 and 3999±5892 copies/cell respectively. By comparison, the CD34+ cells with low levels of surface CD71 in either EPO(+) or EPO(−) cultures demonstrated detectable levels of gamma globin mRNA in only 7 of 145 sorted cells (5%), and beta-globin mRNA in 85 of 145 cells (59%), with total (gamma+beta) globin mRNA under 200 copies/cell in >90% of those cells. These novel results suggest that the frequency and levels of gamma-globin transcripts are quite low at the earliest stages of an EPO response among adult human CD34+ cells. However, it is clearly demonstrated that the cells can increase their capacity to import iron and transcribe beta-globin mRNA at very high levels prior to their first EPO-dependent cell division.

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