Erythroid Differentiation Of CD34 Research Articles

Optimization of β-globin Stable Expression Using the Third Generation Lentiviral Vector for β-thalassemia Therapy

To provide a research basis for a safe and effective cell therapy for β-thalassemia through optimization of HS4 region of the third generation lentiviral vector for stable expression of β-globin. The human β-globin HS4 region in the third generation lentiviral expression vector was optimized to construct the lenti-HBB, and the transcription and translation of β-globin gene were analyzed by RT-PCR and Western blot after the transduction of lenti-HBB in MEL cell line. Furthermore, the erythroid differentiation of CD34+ cells which were transduced lentiviral virus carrying human β-globin from normal human umbilical cord blood cells and peripheral blood cells of patients with β-thalassemia major were confirmed by colony formation assay, cell smear assay and flow cytometry. The safety and effectiveness of the optimized lenti-HBB were verified by NSG mouse in vivo test. The human β-globin was expressed stably in the MEL cells, and CD34+ cells from health umbilical cord blood as well as PBMC from patient with β-thalassemia major transduced with lenti-HBB could be differentiated to mature red blood cells. The β-globin expression and differentiation in CD34+ cells were demonstrated successfully in the NSG mouse for about 35 months after post-transplant. Stable β-globin expression through the optimization of HS4 from CD34+ in the third generation lentiviral vector is safe and effective for patients with severe β-thalassemia and other β-globin abnormal diseases.

The Histone Methyltransferase Setd8 Alters the Chromatin Landscape and Regulates the Expression of Key Transcription Factors during Erythroid Differentiation

Setd8 is the sole methyltransferase capable of mono-methylating histone H4, lysine 20. Setd8 mRNA is expressed ~10-fold higher in erythroid cells than any other cell type (biogps.org) and Setd8 protein levels increase in concert with GATA1 levels during erythroid differentiation of CD34+ HSPCs, suggesting Setd8 may have a role regulating the erythroid transcriptome. Consistent with this hypothesis, erythroid deletion of Setd8 is embryonic lethal by embryonic day 11.5 (E11.5) due to profound anemia and global transcriptomic analyses of sorted populations of E10.5 Sed8 null and control erythroblasts demonstrated a profound defect in transcriptional repression, with 340/345 differentially expressed genes (DEG) expressed at higher levels in the Setd8 null cells than controls (Malik Cell Reports 2017).Primitive erythroblasts mature and enucleate in a semi-synchronous manner in circulation. To better understand the function of Setd8 in regulating the erythroid transcriptome, we extended our transcriptomic analyses by performing RNA-seq in sorted E9.5 Sed8 null (EpoRCre+; Setd8 Δ/Δ) and control (EpoRCre+; Setd8 Δ/+) erythroblasts. The Setd8 null cells failed to repress 20/137 (15%) of the genes that are down regulated in control cells from E9.5 to E10.5. Although relatively few genes were impacted, those genes were enriched for the pathway “Oxidative Stress” (adjusted p-value 0.009) suggesting that Setd8 may regulate specific functions during terminal erythroid maturation.We next compared the DEG in Setd8 null erythroblasts to transcriptomic changes that occur as a cell transcends the hematopoietic hierarchy, gaining lineage specificity while suppressing the multi-lineage transcriptome (GSE14833). A large fraction, 105/345 (~30%), of genes up-regulated in Setd8 null erythroblasts, are also up-regulated in multipotent progenitors compared to proerythroblasts. In contrast, only 16/345 (5%) were also up-regulated in granulocyte-monocyte progenitors suggesting that Setd8 does not repress other lineage restricted signatures. Together, these results suggest that Setd8 regulates repression of the multi-lineage transcriptome during erythroid differentiation from multipotent progenitor cells.To gain insights into how Setd8 regulates the erythroid transcriptome, we performed ATAC-seq (Buenrostro Nature Methods 2013) on sorted populations of erythroblasts from E10.5 Sed8 null and control embryos. Cell number for the Setd8 null samples was limited due to anemia, with ~1000 cells used for each replicate. Setd8 and control replicates were aggregated and accessible regions were identified using MACS2. Regions more accessible in Setd8 null cells were identified by computing a log2 ratio between Setd8 null and control samples using deepTools bamCompare. In addition, we utilized ChIPmentation (Schmidl Nature Methods 2015) to assay H3K27me3 occupancy across the genome of WT E10.5 erythroblasts to identify regions of heterochromatin in maturing erythroblasts. Two replicates were performed using 2.5-5x105 cells per assay, and peak called was done using MACS2.A total of 157 genes were identified that had more accessible chromatin in Setd8 null cells and contained an enrichment for H3K27me3 in WT cells suggesting that these genes should be repressed during normal erythropoiesis. Among these were several DEG that were up-regulated in the Setd8 null cells including Hhex, Cd63, and Gata2. Genomic data integration also identified several additional transcriptional regulators that are active in earlier hematopoietic progenitors but typically silenced during erythroid differentiation including Notch1 and Cebpa. Pathway analysis of the 157 genes identified several stemness-related pathways including “Transcriptional regulation of pluripotent stem cells” and “OCT4, SOX2, NANOG repress genes related to differentiation” (adjusted p-values 0.005 and 0.008, respectively). The chromatin regions that were more accessible in the Setd8 null cells were enriched for the DNA binding motifs of the transcription factor ERG (p-value 1-257), SCL (p-value 1e-193), and NRF1 (p-value 1e-101). Taken together, these data suggest that Setd8 works in concert with erythroid transcription factors to repress the transcriptional network in stem and progenitor cells and establish appropriate patterns of gene expression during erythroid differentiation. DisclosuresNo relevant conflicts of interest to declare.

TET2 is upregulated during erythroid differentiation of CD34+ cells from healthy donors and myelodysplastic syndrome patients

BackgroundTet methylcytosine dioxygenase 2 (TET2) is frequently mutated and/or downregulated in myeloid neoplasm, including myelodysplastic syndromes. Despite the extensive studies, the specific contribution of TET2 in disease phenotype of myeloid neoplasms is not fully elucidated. Recent findings have grown attention on the role of TET2 in normal and malignant erythropoiesis.MethodsIn the present study, we investigated TET2 mRNA levels by quantitative PCR during erythropoietin-induced erythroid differentiation CD34+ cells from healthy donor and myelodysplastic syndrome patients. Statistical analyses were performed using the ANOVA and Bonferroni post hoc test and a p-value <0.05 was considered statically significant.ResultsTET2 expression is upregulated during erythroid differentiation of CD34+ cells from healthy donor and myelodysplastic syndrome patients.ConclusionsOur findings corroborate that TET2 is involved in the erythrocyte differentiation.

Re-Creating Hereditary Persistence of Fetal Hemoglobin (HPFH) to Treat Sickle Cell Disease (SCD) and β-Thalassemia

Hereditary persistence of fetal hemoglobin, a naturally occurring condition that substantially ameliorates disease in SCD and β-thalassemia, is associated with genetic variation at the β-globin locus. Our strategy is to use the CRISPR-Cas9 system to re-create specific genetic variants associated with HPFH in CD34+ hematopoietic stem and progenitor cells (HSPCs) and demonstrate their causal relationship to elevated fetal hemoglobin (HbF) levels as a potential therapeutic strategy to treat SCD and β-thalassemia.The CRISPR-Cas9 system has been adapted to achieve site-directed DNA cleavage by guide RNAs (gRNAs) and the Cas9 endonuclease. We have identified highly potent single gRNAs (sgRNAs) that can target regions within the β-globin locus associated with HPFH. The sgRNAs, when combined and delivered as dual sgRNAs, resulted in deletions that mimic naturally occurring deletions associated with HPFH. We have also optimized transfection dose and ratio of these sgRNAs with either Cas9 mRNA or Cas9 protein into primary human CD34+ HSPCs from mobilized peripheral blood of healthy donors and can achieve greater than 85% transfection rate with minimal cell loss (85-95% viability). We found that sgRNAs delivered with Cas9 protein resulted in better cell viability than when delivered with Cas9 mRNA especially at higher doses, while attaining the same rates of editing efficiency. Lastly, erythroid differentiation of CD34+ HSPCs demonstrated significant increase in γ-globin gene expression level by qRT-PCR as well as HbF protein levels, measured by FACS and LC/MS.In conclusion, we have optimized sgRNA and Cas9 transfection into primary human CD34+ HSPCs. We have identified sgRNAs that are highly effective in targeting the β-globin locus and employed forward genetics to re-create genetic variants associated with HPFH in HPSCs. We have demonstrated the causal relationship of different HPFH genetic variants to elevation of HbF, and obtained comparative data on upregulation of HbF in erythroid cells differentiated from edited CD34+ HSPCs. Our findings provide a viable therapeutic strategy using CRISPR-Cas9 for the treatment of β-hemoglobinopathies. DisclosuresLin:CRISPR Therapeutics: Employment. Paik:CRISPR Therapeutics: Employment. Mishra:CRISPR Therapeutics: Employment. Chou:CRISPR Therapeutics: Employment. Zhang:CRISPR Therapeutics: Employment, Equity Ownership. Tomkinson:CRISPR Therapeutics: Employment. Pettiglio:CRISPR Therapeutics: Employment. Sanginario:CRISPR Therapeutics: Employment. Woo:CRISPR Therapeutics: Employment. Lee:CRISPR Therapeutics: Employment. Allen:CRISPR Therapeutics: Employment. Cradick:CRISPR Therapeutics: Employment. Chakraborty:CRISPR Therapeutics: Employment. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding. Cowan:CRISPR Therapeutics: Employment, Equity Ownership. Novak:CRISPR Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership.

Quantitative Proteomic and Transcriptomic Analysis Reveals Post-Transcriptional Regulation of Mitochondrial Biogenesis during Erythropoiesis

The differentiation and maturation of erythroid cells require highly regulated patterns of gene expression and metabolism. Mitochondria are critical for heme biosynthesis and iron metabolism in erythroid cells, yet their regulation during normal erythroid maturation remains largely unexplored. Here we measured global protein and mRNA expression in primary human fetal liver and adult bone marrow-derived CD34+ hematopoietic stem/progenitor cells (HSPCs) and differentiated erythroid precursors (proerythroblasts or ProEs) by mass-spectrometry-based quantitative proteomics and RNA-seq analysis, respectively. In-depth proteomic profiling resulted in identification and high-quality quantification of proteins encoded by 14,502 genes, accounting for 72.4% of the total annotated protein-coding genes in humans. Through unbiased and comprehensive comparison of the proteomic and transcriptomic dynamics between primary HSPCs and committed erythroid precursors, we discovered a number of previously unrecognized regulatory pathways essential for normal erythropoiesis.Importantly, we identified key pathways related to mitochondrial biogenesis, including ATP biosynthesis, electron transport chain, oxidative phosphorylation, and cellular respiration, whose protein expression was substantially induced during erythropoiesis. Strikingly, the increases in protein expression were not paralleled by changes in mRNA expression of these genes, suggesting that they are regulated through post-transcriptional mechanisms. Consistent with the enhanced mitochondrial protein expression, we observed substantial increases in mitochondrial membrane potential (MMP) and intracellular ATP level during early erythroid differentiation before clearance of mitochondria at terminal maturation stages. We next profiled metabolite levels in HSPCs and erythroid precursors at various differentiation stages, and observed progressive progressive alterations of metabolites from the tricarboxylic acid cycle and other mitochondrial pathways during erythroid maturation. Furthermore, we identified two principal mitochondria-associated transcription factors, mitochondrial transcription factor A (TFAM) and Prohibitin 2 (PHB2), whose protein but not mRNA expression was substantially increased during erythroid maturation. Depletion of TFAM or PHB2 expression markedly impaired erythroid differentiation from primary human CD34+ HSPCs. TFAM or PHB2-deficient cells displayed impaired erythroid differentiation, proliferation and hemoglobin expression, reduced mitochondrial mass, membrane potential and ATP synthesis, and increased apoptosis. Pharmacological inhibition of mitochondrial activities by targeting mitochondrial complex I (metformin, phenformin, and rotenone), complex III (antimycin A), or complex V (oligomycin) in CD34+ HSPCs impaired erythroid differentiation in a dose-dependent manner, consistent with an essential role of mitochondria for normal erythroid development.To elucidate the regulatory mechanisms underlying the developmental control of mitochondrial biogenesis, we measured protein synthesis rate in CD34+ HSPCs and differentiated erythroid cells by analyzing the incorporation of the methionine analogue HPG (L-homopropargylglycine). The rate of HPG incorporation was the lowest in undifferentiated CD34+, and increased by 1.8 and 2.5-fold in CD71+CD235a- and CD71+CD235a+ erythroid progenitor cells, respectively. Consistent with the progressive increase in protein synthesis, we observed a gradual increase of mTORC1 signaling, as measured by the phosphorylated eIF4-E binding protein (4E-BP1) and S6 kinase (p70S6K), during erythroid differentiation of CD34+ HSPCs. Inhibition of mTORC1 signaling by active-site mTOR inhibitors markedly impaired expression of mitochondria-associated proteins, mitochondrial mass and membrane potential, and erythroid differentiation of CD34+ HSPCs. Thus, our results support a model that mitochondrial biogenesis is highly regulated through mTORC1-mediated activation of protein translation during erythroid lineage specification. Our studies also suggest a novel mechanism for proper regulation of mitochondrial biogenesis by post-transcriptional machinery, and may have direct relevance to the hematological defects associated with human mitochondrial diseases and aging. DisclosuresDeBerardinis:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Peloton Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Long-Term Increase in Fetal Hemoglobin Expression in Nonhuman Primates Following Transplantation of Autologous Bcl11a Nuclease-Edited HSCs

Elevated levels of fetal hemoglobin (HbF) ameliorate the clinical symptoms of beta-thalassemia and sickle cell anemia. The transcription factor B-cell lymphoma/leukemia 11A (BCL11A) is required for silencing of gamma-globin expression in adult erythroid cells and functions as a switch from fetal to adult hemoglobin production in humans. BCL11A therefore constitutes a therapeutic target for the treatment of hemoglobinopathies. We inactivated BCL11A function by double-strand DNA break-induced mutagenesis using Transcription Activator-Like Effector Nucleases (TALENs). 20 to 30% gene editing could be achieved in vitro in human and nonhuman primate CD34+ cells by TALEN mRNAs electroporation targeting exon 2 of Bcl11a. Colony-forming efficiency was slightly lower in Bcl11a-edited CD34+ cells but lineage differentiation potential was unchanged. Erythroid differentiation of CD34+ cells in culture showed increased Fetal to Beta hemoglobin ratio in both human and primate Bcl11a-modified cells as compared to control cells, thus validating our editing approach to increase HbF production.To determine if Bcl11a-edited hematopoietic stem cells (HSCs) could be engrafted and give rise to HbF-producing erythrocytes, we transplanted a pigtail macaque with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. We detected about 1 % gene disruption in vivo early post-transplant and disruption frequency gradually declined to reach a set point of about 0.3% starting at day 28 post-transplantation. In this analysis, which we have so far taken out to 42 days, single clones could be tracked based on their mutation signature, and we found that several clones persisted over time, confirming engraftment of Bcl11a-modified cells. Since the transplantation procedure and chemo-radiotherapy conditioning can raise HbF production, three control animals that were transplanted using similar conditions as with the Bcl11a-edited HSCs and one untransplanted animal were also included in our analysis. Flow cytometry measurement of HbF in peripheral blood showed a rapid increase in F-cell production in all animals, reaching levels that ranged from 10% to 40% by 30 days, while the untransplanted control showed basal HbF expression of about 0.5% (Fig. 1A). The peak for HbF expression lasted for about 140 days and eventually returned to basal levels that averaged 0.5% for all control animals. In comparison, the animal transplanted with Bcl11a-edited cells showed significantly higher HbF levels starting at day 140 post-treatment (1-1.5%), and HbF production has remained constant for at least 150 days. This result was confirmed by hemoglobin mRNA analysis in peripheral blood using real-time PCR. We found a rapid increase in gamma globin expression following transplantation, before returning to near basal levels. As compared to controls, the animal transplanted with Bcl11a-edited cells showed a 5 to 10-fold increase in gamma to beta globin ratio at day 140 and this ratio has remained constant ever since (Fig. 1B). We are currently working on ways to enhance Bcl11a-editing and to select for Bcl11a-modified HSCs using targeted integration of the chemoselection cassette P140K MGMT to ultimately achieve curative HbF production. Potential TALEN off-target sites will also be examined as well as any side effect associated with the inactivation of BCL11A. Overall, our data demonstrate that transplantation of Bcl11a-edited HSCs results in elevated HbF production in nonhuman primates. Furthermore, we show that nonhuman primates can serve as a useful model for novel gene editing strategies toward the treatment of hemoglobinopathies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Distinct Roles of TET Proteins in the Regulation of Normal and Disordered Human Erythropoiesis

TET family proteins (TET1, TET2 and TET3) have recently emerged as important epigenetic modifiers by catalyzing the conversion of 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc). Although they have been documented to play important roles in a variety of biological processes, their function in erythroid differentiation has yet to be defined. In the present study, we show that of the three TET family members, TET2 and TET3 but not TET1 are expressed in erythroid cells and that TET3 is more abundantly expressed than TET2. Using shRNA-mediated knockdown approach we explored the role of TET proteins in erythroid differentiation of CD34+ human cells. We first showed that consistent with their role in the production of 5hmc, knockdown of either TET2 or TET3 led to a decrease in global 5hmc levels as assessed by mass spectrometric analysis. However, knockdown of TET2 or TET3 resulted in distinctly different phenotypic changes during erythropoiesis. Knockdown of TET3 in human CD34+ cells resulted in impaired cell growth which is accompanied by increased apoptosis of late stage erythroblasts. Knockdown of TET3 also led to generation of bi/multinucleated polychromatic/orthochromatic erythroblasts which is accompanied by impaired enucleation. To explore the molecular mechanisms for the observed phenotypic changes, we performed RNA-seq analysis on control and TET3 knockdown erythroblasts at same stages of development. Bioinformatics analysis revealed that the expression levels of several apoptosis-promoting genes such as FOXO1, TNFRSF10B, TGFB1 and BTG1 are increased and that of a mitosis/cytokinesis associated gene KLHDC8B is decreased in polychromatic and orthochromatic erythroblasts following TET3 knockdown. Measurement of 5hmc and 5mc at promoter region of KLHDC8B locus revealed decreased 5hmc level concurrent with increased 5mc level. Importantly, knockdown of KLHDC8B in CD34+ cells as with knockdown on TET3 led to generation of increased numbers of bi/multinucleated polychromatic/orthochromatic erythroblasts and impaired enucleation implying a role for this protein in cytokinesis of late stage but not early stage erythroblasts. These findings demonstrate that TET3 regulates erythropoiesis in a stage-specific manner by targeting different set of genes. Importantly, knockdown of TET2 led to phenotypic changes that were very different from that seen following knockdown of TET3 but the observed changes are similar to the erythroid development defects noted in myelodysplastic syndromes (MDS). These include hyper-proliferation of early stage erythroid cells; delayed terminal erythroid differentiation and increased apoptosis of late stage erythroblasts. Together with the fact that TET2 gene mutation is one of the most common mutations in MDS and dyserythropoiesis is a hallmark of this disorder, our findings suggest that TET2 gene mutations can directly account for dyserythropoiesis of MDS. Our findings demonstrate distinct and important roles for TET2 and TET3 in regulating erythropoiesis and provide significant new and novel insights into epigenetic regulation of erythropoiesis at distinct development stages. The findings are likely to be very useful for furthering our understanding of epigenetic regulation of normal and disordered human erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

A Candidate Trans-Acting Modulator of Fetal Hemoglobin Gene Expression in the Arab-Indian Haplotype of Sickle Cell Anemia

In the Arab-Indian (AI) β-globin gene (HBB) haplotype of sickle cell anemia, fetal hemoglobin (HbF) levels are higher and the disease phenotype milder than in African HBB haplotypes. To study the genetic basis of HbF regulation in the AI haplotype, whole genome sequencing (WGS) was done in 14 unrelated Saudi AI haplotype HbS homozygotes; 7 selected for low HbF (7.9±1.9%) and 7 for high HbF (18.5±3.2%). WGS was also done in 3 Indians with the AI haplotype (HbF 26.0±4.5%), 3 African Americans with the Benin haplotype selected because of the uncommonly high HbF for this haplotype (19.8±0.4%) and 1 African American homozygote with the Senegal haplotype (HbF 16.0%). Association of SNPs with HbF was tested using simple linear regression with an additive genetic model, and the Sequence Kernel Association Test using sliding windows of 5 SNPs. We found 465 SNPs that were significantly associated with HbF in a single SNP analysis (p≤10-5). Following annotation using SNPper and ENCODE RegulomeDB, PLINK was employed to remove all SNPs in high linkage disequilibrium (r2>0.8) ending with 128 SNPs for follow-up analysis. These prioritized variants were further investigated using functional evaluation tools such as SCAN, Hembase, ErythronDB and the results of WGS were followed by genome-wide SNP analysis, imputation of genotypes to 1000genomes, and targeted direct genotyping in the following cohorts with sickle cell anemia: 110 AI haplotype cases (HbF 18.0±7.0%); 71 Saudi Benin haplotype cases (HbF 11.4±4.1%); 44 Indian AI haplotype homozygotes (HbF 23.0±4.8%); 894 African Americans with diverse HBB haplotypes (HbF 5.2±5.6%). All cases were examined when the HbF levels had stabilized. None of the patients were using hydroxyurea when HbF was measured. Based on WGS, 2 SNPs, rs4527238 and rs35685045 (D'=1) in intron 9 of ANTXR1 (anthrax toxin receptor 1, 2p13.1) remained associated with HbF after correction for multiple comparisons. This result was replicated in the cohort of 110 additional AI haplotype Saudi patients but not in cohorts with other HBB haplotypes. The alternative alleles of these 2 SNPs had nearly equal frequencies in Saudis with the Saudi AI haplotype and lower frequencies in Indians, Saudi Benin haplotype and African Americans with sickle cell anemia. The T allele of rs4527238 and the C allele of rs35685045 are associated with high HbF. ANTXR1 SNPs explained 10% of the HbF variability compared with 8% for BCL11A; these genes had independent, additive effects on HbF and explained about 15% of HbF variability. RNA sequencing and gene expression microarray analysis at 3 time points during the erythroid differentiation of CD34+ and iPSCs isolated from the same subjects showed that only the long splice variant of ANTXR1 was expressed. Expression increased over time in CD34+ erythroid cells and decreased over time in iPSC-derived erythroid progenitors in a pattern similar to BCL11A expression, with increasing expression as CD34+ cells matured and HbF expression fell and declining expression as iPSCs differentiated and HbF expression increased. Immunofluorescence co-staining for ANTXR1 and HbF during the erythroid differentiation of sickle iPSCs showed that reduced expression of ANTXR1 was paralleled by increased expression of HbF. By GTEx analysis, eQTLs at rs4527238 and rs35685045 showed a trend for increased expression according to genotype with rs4527238 T/T having the lowest level of expression and T/C and C/C genotypes, higher expression levels (p= 0.08) and for rs35685045, C/C the lowest expression levels and C/T and T/T higher expression levels (p= 0.07); rs4527238 and rs35685045 had no effect on the expression of BCL11A, a repressor of HbF expression located at 2p16 (p=0.8). STRING 9.1 predicted protein interactions of ANTXR1 directly with LRP6, HDAC2, BCLX and BRAC1 with the highest level of confidence (0.95) with LRP6. The HbF phenotype in AI haplotype sickle cell anemia is unique and ANTXR1 is the first trans-acting element associated with HbF whose effects appear to be limited to the Saudi AI haplotype. ANTXR1 might have dual effects on HbF, indirectly affecting hematopoiesis via interactions with LRP6 and Wnt signaling, and directly modulating HBG expression through its effects on HDAC2. A dual effect on HbF expression has been suggested for MYB. Whether targeting ANTXR1 is a therapeutic option will require further studies. DisclosuresNo relevant conflicts of interest to declare.

MASL1 induces erythroid differentiation in human erythropoietin-dependent CD34+ cells through the Raf/MEK/ERK pathway

AbstractHuman erythropoiesis is a dynamic and complex multistep process involving differentiation of early erythroid progenitors into enucleated RBCs. The mechanisms underlying erythropoiesis still remain incompletely understood. We previously demonstrated that erythropoietin-stimulated clone-1, which is selectively expressed in normal human erythroid-lineage cells, shares 99.5% identity with malignant fibrous histiocytoma-amplified sequences with leucine-rich tandem repeats 1 (MASL1). In this study, we hypothesized that the MASL1 gene plays a role in erythroid differentiation, and used a human erythroid cell culture system to explore this concept. MASL1 mRNA and protein expression levels were significantly increased during the erythroid differentiation of CD34+ cells following erythropoietin (EPO) treatment. Conversely, MASL1 knockdown reduced erythroid differentiation in EPO-treated CD34+ cells. In addition, MASL1 knockdown interrupted the Raf/MEK/ERK signaling pathway in CD34+ cells. MASL1 mutant-transfected CD34+ cells also showed decreased erythroid differentiation. Furthermore, inhibition of the SH3 domain of Son of Sevenless, which is an upstream adapter protein in EPO-induced erythroid differentiation, also reduced MASL1 expression and phosphorylation of Raf/MEK/ERK kinases that consequently reduced erythroid differentiation of EPO-induced CD34+ cells. Importantly, we also demonstrated that MASL1 interacts physically with Raf1. Taken together, our data provide novel insights into MASL1 regulation of erythropoiesis through the Raf/MEK/ERK pathway.

Missense Mutations in the ABCB6 Transporter Cause Dominant Familial Pseudohyperkalemia

AbstractAbstract 3184Isolated Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by cold-induced slow ‘passive leak' of red cell K+ into plasma, first described in a large Scottish family from Edinburgh (Stewart GW, et al., 1979). Although in freshly obtained blood samples plasma [K+] was normal, it was increased when measured in blood stored at or below room temperature. This trait was unaccompanied by clinical symptoms or signs except for mild abnormalities of red cell shape. FP Lille was later described in a large Flemish kindred with morphologically normal red cells (Dagher G, et al., 1989; Vantyghem MC, et al., 1991). In this family, red cell K+ efflux measured in the presence of ouabain and bumetanide was normal at 37°C, but greatly increased at 22°C and 9°C. FP Lille mapped to 2q35-q36 (Carella M, et al., 2004), whereas FP Edinburgh mapped to 16q23-qter (Iolascon A, et al., 1999). Subsequently, asymptomatic cases FP Chiswick and FP Falkirk with remarkable increased MCV were reported (Haines PG, et al., 2001).Functional gene mapping and sequencing analysis of the candidate genes within the 2q35-q36 critical interval in three multigenerational FP families with 20 affected individuals identified two novel heterozygous missense mutations in the ABCB6 gene that cosegregated with disease phenotype. The two genomic substitutions altered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that in erythrocyte membranes bears the Langereis blood group antigen system (Krishnamurthy PC, et al., 2006; Helias V, et al., 2012). Structural modeling predicts subtle changes in protein structure associated with either mutation.ABCB6 mRNA and protein levels increased during erythroid differentiation of CD34+ erythroid precursors (at 7 and 14 days of EPO induced differentiation), and of HEL and K562 erythroleukemia cells. However, the ABCB6 R375Q mutation altered neither levels of ABCB6 mRNA or protein, nor protein localization in mature erythrocytes or erythroid precursor cells. These data strongly suggest that missense mutations in residue 375 of the ABCB6 polypeptide either mediate the cold-induced K+ leak of chromosome 2-linked FP, or activate an independent, cold-induced cation permeability pathway of the red cell. Disclosures:No relevant conflicts of interest to declare.

Downregulation of IRS2 in myelodysplastic syndrome: A possible role in impaired hematopoietic cell differentiation

Insulin receptor substrate 2 (IRS2) is an adaptor protein that associates with the receptor of erythropoietin, insulin-like growth factor 1 and thrombopoietin; however, its role is not known in myelodysplasia. We, herein, report a significantly lower IRS2 expression in MDS cells, compared to normal cells. IRS2 expression was reduced in high-risk, compared to low-risk disease, and positively correlated with neutrophil and platelet counts. IRS2 was upregulated during erythroid differentiation of CD34+ cells from normal donors and low-risk MDS patients and also during erythroid, granulocytic and megakaryocytic differentiation in cell lines. These results suggest that defective IRS2 expression plays a role in the impaired hematopoietic cell differentiation in MDS.

MASL1 Induces Erythroid Differentiation In Human Erythropoietin-Dependent CD34+ Cells Involving Raf/MEK/ERK Signaling Pathway

AbstractAbstract 4228Human erythropoiesis is a dynamic complex multistep process that involves differentiation of early erythroid progenitors to enucleated red blood cells. Importantly, erythroid differentiation involves lineage-specific activation and restriction of gene expression. However, the mechanisms that play a role in erythropoiesis still remain incompletely understood. We previously demonstrated that erythropoietin-stimulated clone-1 (EP1), which is selectively expressed in normal human erythroid lineage cells shares 99.5% identity with the malignant fibrous histiocytoma amplified sequence 1(MFHAS1 or MASL1). MASL1 is an important oncogene that is highly expressed in malignant fibrous histiocytomas (MFH). MASL1 protein has several domains; ras, three leucine zipper, ATP/GTP-binding site and leucine-rich tandem repeat motif which are important structural or functional elements for interactions among proteins related to the cell cycle. However, the function of the MASL1 gene in erythropoiesis has not been studied. We hypothesized that MASL1 gene plays a role in the erythroid differentiation, and used a human liquid erythroid culture system to explore this concept. MASL1 mRNA and protein expression levels were significantly increased during the erythroid differentiation of CD34+ cells following EPO treatment. Conversely, small interfering RNA-mediated knock down of MASL1 in CD34+ cells resulted in a reduction of a double positive of transferrin receptor and glycophorin A (GPA) (14.1 ± 4.7%) when compared with mock (77.9 ± 4.4%) or control vector-transfected CD34+ cells (76.7 ± 8.8%) at day 14. May-Grunwald-Giemsa staining confirmed a phenotype of MASL1 knockdown CD34+ cells that most of cells were pro- or basophilic erythroblasts. Western blotting also showed a significant decrease in hemoglobin protein levels in MASL1 knockdown CD34+ cells. In addition, MASL1-knockdown in CD34+ cells demonstrated an interruption of Raf/MEK/ERK signaling pathway. Inhibition assay of SH3 domain of Son of Sevenless (SOS) which is an upstream adapter protein in EPO-induced erythroid differentiation, confirmed that a suppression of MASL1 reduced the phosphorylation of Raf/MEK/ERK kinases and delays erythroid differentiation of Epo-induced CD34+ cells. Taken together, our study provides novel insights into MASL1-regulated erythropoiesis through the Raf/MEK/ERK signaling pathway. Disclosures:No relevant conflicts of interest to declare.

IRS2 Is Dowregulated In Primary MDS Cells and During MDS Erythroid Differentiation

AbstractAbstract 1886Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress towards acute myeloid leukemia (AML). The abnormal differentiation of myeloid and erythroid cells is probably involved in the pathogenesis of MDS. Insulin receptor substrates (IRS) are adaptor proteins that link signaling, from upstream activators, to downstream effectors to modulate normal growth, metabolism, survival and differentiation. IRS2, a member of the IRS family, binds to Insulin Grow Factor 1 receptor (IGF1R) and Erythropoietin receptor (EPOR). It is upregulated and phosphorylated by EPO in normal bone marrow erythroblasts and in UT-7 leukemic cells, as well as in HL60 leukemic cells during granulocytic-differentiation upon DMSO induction. IGF1 signaling is capable of inducing differentiation in several cell types and plays an important role in the regulation of human erythropoiesis. EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. In view of the role of IRS2 in the erythroid and granulocytic differentiation process, we hypothesized that IRS2 might be related to the deficient differentiation of MDS cells and disease progression. The aim of the present study was to characterize the mRNA and protein expression levels of IRS2 in cells of MDS patients and normal donors and to analyze the IRS2 expression levels between low-risk and high-risk of MDS. We also elucidated the expression levels of IRS2 transcripts during erythroid differentiation of CD34+ cells from normal donors and MDS patients. We studied 12 healthy donors and 29 patients with MDS at the time of diagnosis (16 low-risk [RA/RARS] and 13 high-risk [RAEB/RAEBt] according to FAB classification; 15 low-risk [RCUD/RCMD/RARS] and 11 high-risk [RAEB-1/RAEB-2] according to WHO classification; 22 low/INT-1 risk and 7 INT-2/high risk according to IPSS; 25 low risk and 4 intermediate/high risk cytogenetic). RT-PCR was performed in total cell from bone marrow samples for gene expression studies. Protein expression was evaluated by Western blot in bone marrow mononuclear cells from MDS patients or in peripheral blood CD34+ from normal donors. Erythroid-differentiation was performed in CD34+ bone marrow cells from 4 normal donors and 4 MDS patients. IRS2 gene expression was significantly decreased in primary MDS cells compared with normal cells (0.74 [4.06-0.15] vs. 4.71 [11.78-0.66], P<0.0001). Western blot analysis corroborated these findings. According to FAB and WHO classifications, real time RT-PCR demonstrated a significantly lower expression of IRS2 in high-risk MDS samples when compared with low-risk: FAB, 0.34 [0.15–1.56] vs. 1.05 [0.19–4.06], P=0.0172; WHO, 0.30 [0.15–1.44] vs. 1.00 [0.19–4.06], P=0.0204. Based on the IPSS classification and cytogenetic risk group, the expression levels of IRS2 were similar between the low and high-risk groups. During erythroid differentiation, we evaluated IRS2 gene expression of CD34+ cells on days 6, 8 and 12 of culture. On day 12 of normal CD34+ erythroid differentiation, there was an 8.25-fold increased in IRS2 expression, compared to day 6. Interesting, MDS CD34+ cells showed a lower increment in IRS2 transcripts at the same time point (3.89-fold increase only). In conclusion, the down regulation of IRS2 in primary MDS cells and the lower increase in its expression during MDS erythroid differentiation, suggest a role of IRS2 to maintain the effective hematopoiesis. Moreover, IRS2 lower expression in high-risk group, suggests that IRS2 plays a role in the MDS pathophysiology and disease progression. Supported by FAPESP and CNPq. Disclosures:No relevant conflicts of interest to declare.

CTD small phosphatase like 2 (CTDSPL2) can increase ε- and γ-globin gene expression in K562 cells and CD34+ cells derived from umbilical cord blood

BackgroundA potential strategy for treatment of sickle cell disease (SCD) and β-thalassemia in adults is reactivation of the ε- and γ-globin genes in the adult. We aimed to identify trans-activators of ε- and γ-globin expression and provide new candidate targets for effective treatment of sickle cell disease (SCD) and β-thalassemia through activation of ε- and γ-globin genes in adults.ResultsWe identified a CTD small phosphatase like 2 (CTDSPL2) gene that had higher transcription levels in umbilical cord blood (UCB) than in adult bone marrow (BM). Also, transcription of the CTDSPL2 gene increased significantly during erythroid differentiation. Further, we found that overexpression of CTDSPL2 could obviously improve the expression of ε- and γ-globin genes in K562 cells. Meanwhile, the repression of CTDSPL2 by RNA interference decreased expression of ε- and γ-globin genes but did not inhibit the increase of globin gene expression during K562 erythroid differentiation. In addition, the enforced expression of CTDSPL2 gene mediated by lentiviruses could also increase ε- and γ-globin gene expression during erythroid differentiation of CD34+ cells derived from UCB.ConclusionCTDSPL2 gene can obviously improve the expression of ε- and γ-globin genes in K562 cells and CD34+ cells derived from UCB. Our study provides a new candidate target for effective treatment of SCD and β-thalassemia.

Deranged MicroRNA 16-2 Expression Contributes to Erythropoiesis in Polycythemia Vera.

Abstract 3896Poster Board III-832We previously reported the finding of significantly raised levels of mature microRNA 16 (miR-16) in CD34+ cells from PV patients (pts) (Guglielmelli P et al, ASH 2008, 179A). Mature miR-16 can derive from both miR-16-1 at chr13q14.3 and miR-16-2 at chr3q26.1, which differ in their precursor miRNA (pre-miR) sequence. To distinguish between either genes as a source of increased miR-16 levels we quantified each pre-miR in PV CD34+; we observed that the premiR-16-1/16-2 ratio decreased from 1.47± 2.38 in controls (ctr) to 0.52± 0.34 in PV (P<.001). To confirm the preferential expression of miR-16-2 we knocked-down miR-16-1 or miR-16-2 using specific siRNAs; in PV CD34+ treated with miR16-2 siRNA the miR-16 levels were 90±9% lower than in scramble-treated cells while miR16-1 siRNA caused only a 10±3% reduction (P <.01). Levels of miR-16 in PV CD34+ were not correlated to the JAK2V671F burden; also, there was no modification of miR-16 in JAK2V617F mutant HEL or UKE-1 cells after treatment with the JAK2 inhibitor AZD1480. These data suggested that the abnormally increased mature miR-16 in PV are largely derived from miR-16-2. To address underlying mechanisms we performed direct sequencing at the miR-16-1 and miR-16-2 regions without detecting sequence abnormalities; also we excluded gene copy number changes (SNP 6.0 array). The promoter of miR16-2 is not characterized yet, but recent data suggest that it might be the same of the structural maintenance of chromosomes-4 (SMC4) gene, to which miR16-2 is intronic. We have found a significant correlation between miR-16 and SMC4 mRNA levels in PV CD34+ (R=0.77, P<.001); furthermore, after induced erythroid differentiation of normal CD34+ a concurrent increase of miR-16 and SMC4 mRNA was observed, indirectly supporting a shared regulatory control. Methylation-specific PCR and sequencing at upstream CpG-rich regions discovered that PV patients had unmethylated CpGs compared to control cells, suggesting that methylation may concur to miR-16-2 regulated expression. To evaluate involvement of miR16 in abnormal erythropoiesis of PV, we first analyzed the kinetics of miR-16 during erythroid differentiation of CD34+. Levels of miR-16 steadily increased from day 6, when progenitors were switched from proliferative to differentiative phase, up to day 12-14 and, at any time point considered, those measured in PV cells were significantly greater than in ctrs (P= .01). Also, a time-dependent increase of miR16 paralleling the transcription of beta-globin mRNA was observed in hydroxyurea or Na-butyrate treated K562 cells. Then we over-expressed (Amaxa) mature miR16 in normal CD34+ at day 6 of culture, and found that the percentage of CD36+/GPA+ cells increased from 13.6±4.8% in scramble to 28.8±11.3% (P<.05); notably, even in the absence of EPO, some GPA+ cells were generated from miR-16-transduced CD34+. Furthermore, normal CD34+ cells transfected with miR-16 produced significantly increased number of CFU-e and BFU-E (174±100 and 1283±250/103 CD34+) compared to scramble cells (105±90 and 733±90; P<.01) while myeloid colonies were unchanged. A genome-wide expression profiling in normal CD34+ transfected with miR-16-2 siRNA discovered 618 genes significantly de-regulated, among which genes involved in erythroid differentiation such as NFE2, MYB, FGFR2, HLF, GATA-1 and globin family genes (HBA1, HBA2, HBB). Overall, these data support a role of miR-16 in normal erythropoiesis. In case of PV we employed a knock-down strategy using either pre-miR-16-1 or pre-miR-16-2 siRNAs. The use of miR-16-2 siRNA resulted in significant reduction of Epo-independent erythroid colonies (EEC) from GFP-sorted CD34+ cells (60±18% lower than scramble) while no change was observed in cell treated with anti-miR-16-1. These data were confirmed in independent experiments using peripheral blood mononuclear cells (MNC): the number of EEC decreased from 207±20 to 170±23 to 95±25/105 MNC in cells transduced with scramble, anti miR-16-1 or miR-16-2, respectively (P<.01 for miR-16-2 vs others). Also the number of BFU-E generated in cytokine-supplemented cultures of PV MNC was significantly reduced by miR-16-2 siRNA (360±80) compared to cells transduced with scramble (540±120) or miR-16-1 (500±87) (P<.001). In summary, these data support a role for deregulated miR-16-2 in abnormal erythropoiesis of PV, and anticipate its possible relevance as a therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Dynamics of α-globin locus chromatin structure and gene expression during erythroid differentiation of human CD34 + cells in culture

The aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34(+) cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the alpha-globin locus in the developing erythron. A basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34(+) cells. Expression patterns of the alpha- and beta-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the alpha-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis. Human CD34(+) cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the alpha-globin locus during erythroid differentiation of CD34(+) cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the alpha-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and theta gene, and were conspicuously low at the alpha-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the alpha-globin locus as CD34(+) cells differentiated into erythroid pathway. Our results indicate that remodeling of the upstream elements may be the primary event in activation of alpha-globin gene expression. Activation of alpha-globin genes upon EPO treatment involves initial binding of Pol II, downregulation of pre-existing factors like NF-E2, removal of CTCF from the locus, then rebinding of CTCF in an altered pattern, and concurrent or subsequent binding of transcription factors like GATA-1.

A Constitutive Increase in Gamma Globin Gene Production during the Erythroid Differentiation of CD34+ Cells from Sickle Cell Disease Patients and Alterations in Cyclic Nucleotide Levels during This Differentiation.

Studies have demonstrated that the activation of soluble guanylate cyclase (sGC) in erythrocytic cell lines and in primary erythroid precursor culture causes an increase in gamma globin gene transcription and, consequently, an augmented production of fetal hemoglobin (HbF). The cyclic nucleotides, cGMP and cAMP, are second messengers that are responsible for mediating the signaling of various extracellular signals, including those of nitric oxide (NO), hormones and neurotransmitters and, probably, have an important role in SCD by regulating the production of HbF. The aim of this study was to compare gamma-globin gene production, in association with cGMP- and cAMP-dependent signaling pathways, during the erythroid differentiation of CD34+ cells from steady-state SCD patients and healthy controls. Hematopoietic CD34+ cells were separated from the peripheral blood of subjects by CD34+-positive cell magnetic separation and were then cultured on semi-solid medium for 7 days until reaching the erythroid colony forming unit (CFU-e) stage; these colonies were then differentiated in liquid-phase culture until day 13 when cells reached the orthochromatic erythroblast/ reticulocyte stage. Cell samples were collected at days 7, 10 and 13 of culture for flow cytometry, citospin, measurement of intracellular cGMP/cAMP levels and evaluation of the gene expressions of gamma globin and the sGC alpha and beta subunits by real-time RT-PCR. Genes for endothelial nitric oxide synthase (eNOS) and phosphodiesterase 3B (PDE3B) were also studied, since these genes are important for NO/cyclic nucleotide signaling pathways. Gamma globin gene expression was observed to be significantly higher in the cultures of SCD cells on the 13th day of erythroid differentiation compared to the same time point in control cells (2.95 ± 0.30 vs. 1.45 ± 0.17 rel expression, respectively, p<0.01, Mann-Whitney, n=6). The expressions of the sGC alpha and beta subunits, eNOS and PDE3B did not present any significant differences during control and SCD erythroid differentiation. Levels of cGMP were significantly decreased during erythropoiesis in the SCD cultures, compared to control cultures (day 7: 277 ± 53 vs. 639 ± 122 fmol/5x106cells (p<0.05); day 10: 117 ± 22 vs. 268 ± 118 fmol/5x106cells; day 13: 37 ± 6 vs. 93 ± 24 fmol/5x106cells (p<0.02), respectively). In contrast, levels of cAMP were significantly increased in the SCD cultures compared to the control cultures (day 7: 72.02 ± 14.85 vs. 24.99 ± 13.50 pmol/5x106cells (p<0.05); day 10: 36.34 ± 16.71 vs. 8.66 ± 4.59 pmol/5x106cells; day 13: 1.74 ± 1.09 vs. 0.68 ± 0.37 pmol/5x106cells, respectively). Results suggest that the hematopoietic stem cells of SCD patients are significantly and constitutively different to those of control stem cells, since under the same in vitro culture conditions, consistently higher levels of gamma globin were observed during sickle cell erythroid differentiation. Intracellular cGMP and cAMP levels were also significantly altered during SCD erythroid differentiation, providing further support to the theory that cyclic nucleotide signaling may regulate HbF production. The precise role of these signaling pathways in the mechanism studied and whether any cross talk between these pathways occurs remains to be determined.

Defective Expression of ANKHD1 Transcript Variant 3 in Myelodysplastic Cells and Its Role in Apoptosis.

Ineffective erythropoiesis is a common feature of myelodysplastic syndromes (MDS). GATA-1, a transcription factor essential for erythroid maturation was found to be upregulated in normal hematopoietic cells after erythroid differentiation, but not in differentiating MDS erythroblasts. ANKHD1 transcript variant 3, also named PP2500, (NM_024668), is a recently described gene that contains a GATA-1 binding site at its promoter region, and has been shown to be upregulated during erythroid differentiation of normal hematopoietic cells. We then sought to evaluate the expression of PP2500 in normal and MDS cells, the expression of GATA-1 and PP2500 in CD34+ normal and MDS cells during erythroid differentiation, as well as PP2500 functional consequences in terms of apoptosis. Bone marrow samples were obtained from 16 patients with a diagnosis of low-risk MDS (13 RA and 3 RARS) and 5 normal donors, and submitted to RNA extraction. For erythroid differentiation, bone marrow samples from one low-risk MDS patient and one normal donor were submitted to CD34+ selection using MIDI-MACS immunoaffinity columns and cells were plated on plastic culture dishes in methylcellulose medium with appropriate growth factors for 6 days. BFU-E, CFU-E and proerythroblasts were then cultured in alpha MEM for another 8 days. At days 6 and 14, cells were collected and submitted to RNA extraction and apoptosis analysis. Posttranscriptional PP2500 gene silencing was performed in Jurkat cells, through electroporation, using small interfering RNA (SMARTpool siRNA duplexes-Dharmacon). Forty-eight hours after transfection, cells were submitted to RNA extraction and apoptosis analysis. The expression level of mRNA was detected by real time RT-PCR. Endogenous controls used were GAPDH and b-actin. The relative quantification value of gene expression was calculated using 2−DDCT. Apoptosis was evaluated using annexin V-FITC/PI staining; erythroblast differentiation was verified with transferring-receptor/glycophorin-A and FACS analysis. Reduced expression of PP2500 was observed in low-risk MDS cells (RA/RARS) when compared with normal hematopoietic cells (mean ± SD; 0.15 ± 0.18 vs 1.07 ± 0.53, respectively; P=0.0004). Erythroid differentiation of CD34+ normal cells resulted in upregulation of PP2500 and GATA-1: 12-fold increase on day 14 compared to day 6, for both mRNAs. However, differentiating MDS erythroblasts failed to show a noteworthy increment in PP2500 and GATA-1 expressions: 1.5-fold increase for GATA-1 and 2-fold increase for PP2500, on day 14 compared to day 6. After erythroid differentiation, apoptotic cells accounted for 5.6% of cultured normal marrow samples on day 6 and 2.5% on day 14, whereas MDS cultures contained 4.8% apoptotic cells on day 6 and 9.5% on day 14. Posttranscriptional PP2500 gene silencing resulted in a 2-fold increase in apoptotic cells compared to control electroporated cells (27% vs 14% annexin V+/PI−, respectively). In conclusion, reduced expression of PP2500 mRNA was found in low-risk MDS cells compared with normal hematopoietic cells and MDS erythroblasts showed a defective GATA1 and PP2500 expression pattern during erythroid differentiation associated with an increased apoptosis. In addition, PP2500 gene silencing resulted in increasing apoptosis. These results suggest that PP2500 may have an anti-apoptotic function and a defective expression in MDS cells, which may be implicated in the pathophysiological process of MDS.

Delta-4 expression on a stromal cell line is augmented by interleukin-6 via STAT3 activation

Notch receptors and their ligands are known to play an important role in hematopoietic cell fate decisions. Although expressions of Notch ligands were frequently detected in bone marrow cells or thymic epithelial cells, regulatory roles of hematopoietic cytokines on their expression are still poorly understood. In this study, we focused on a new member of Notch ligand family, Delta-4, and analyzed regulatory mechanisms of Delta-4 expression by cytokines using stromal cell lines. For our expression study, we selected a highly Delta-4-expressing murine stromal cell line, SC9-19. Delta-4 protein expression was analyzed by Western blotting after cytokine treatment with or without various kinase inhibitors. Biologic relevance of the enhanced Delta-4 expression was examined in the coculture system using cord blood CD34(+) cells. When SC9-19 was treated with different cytokines, we found interleukin-6 (IL-6) was the most efficient inducer of Delta-4 protein expression. Further analysis revealed that IL-6-induced signaling for Delta-4 expression was transduced through the pathway of Janus kinase/signal transducers and activators of transcription-3 (JAK/STAT3). Other mediators such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and Src tyrosine kinase were not involved in IL-6-induced Delta-4 expression. Erythroid differentiation of CD34(+) cells was enhanced by IL-6 treatment of the Delta-4-expressing original stromal cell line but not of a Delta-4-knockdown stromal cell line. IL-6 augments Delta-4 expression in the stromal cell line via STAT3 activation. This study provides a novel mechanism for augmentation of Delta-4 expression by hematopoietic cytokine and suggests a role for Delta-4 in the control of hematopoiesis.

Identification by differential display of transcripts regulated during hematopoietic differentiation.

The polymerase chain reaction-based differential display method (DDRT-PCR) was used to identify mRNAs differentially expressed during the maturation of human CD34+ progenitor cells stimulated to differentiate in vitro towards granulomonocytic or erythroid lineages with a mixture of hemopoietins (kit ligand + interleukin 3 + GM-CSF in the absence or presence of erythropoietin, respectively). Three cDNA transcripts (B32, B41, and B56) display differential expression during cytokine-induced maturation of CD34+ cells. These clones have no homology with already-described sequences. Primer extension cofirmed the presence of the corresponding mRNA. The levels of mRNA corresponding to B32 are enhanced in the later phases of the granulomonocytic as well as in the erythroid differentiation of CD34+ cells. The mRNA identified by B41 was induced by a late stage in only granulomonocytic differentiation of CD34+ cells. The mRNA corresponding to B56 was instead present in nonstimulated CD34+ cells, declined in the early stages of differentiation, and reappeared at later stages in cells treated with both combinations of cytokines. Expression of these genes was detected in a number of acute myelogenous leukemias, as well as in some leukemic cell lines. B32 and B41 were downregulated in KG-1 cells induced to differentiate towards the monocytic lineage, whereas the levels of B56 were unchanged. In K562 cells, clones B41 and B56 were downregulated only in the late phases of PMA-induced megakaryocytic differentiation and during erythroid differentiation. B32 was rapidly downregulated when K562 cells were induced to differentiate towards either megakaryocytic or erythroid phenotypes. These transcripts represent novel hematopoietic cDNAs that should prove of value for the study of human blood cells and their disorders.