More Efficient Generation of β-Globin-Expressing Erythroid Cells Using Stromal Cell-Derived Induced Pluripotent Stem Cells

Abstract Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. Using traditional embryoid body methods, iPS cell-derived erythroid cells predominantly produce ε-globin and γ-globin without β-globin expression. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with β-globin production, which is associated with emergence of CD34+ hematopoietic stem/progenitor cells (HSPCs) (2014 ASH). In the current study, we extend this work to evaluate erythroid cell generation using iPS cell lines generated from various sources including patients with sickle cell disease (SCD). To test our two hypotheses; (1) erythroid progenitor (EP)-derived iPS cells more efficiently differentiate to erythroid cells, and (2) stromal cell (ST)-derived iPS cells more efficiently emerge hemangioblast-like immature HSPCs which results in greater erythroid cell generation, we generated several clones of iPS cells which were derived from (1) EPs (6 clones) which were differentiated from peripheral blood mononuclear cells and (2) bone marrow STs (5 clones) in SCD patients. Transgene-free iPS cells were generated and characterized according to Merling et al. (Blood. 2013). These iPS cells and controls (2 clones of fibroblast (FB)-derived iPS cells and H1 ES cells) were used to generate ES/iPS sacs for 15 days. After a 2 day culture of ES/iPS sac-derived spherical cells on OP9 feeder cells, the suspension cells were differentiated into erythroid cells using human erythroid massive amplification culture for 13 days (Blood cells Mol Dis. 2002). Following ES/iPS sac generation, 3.5-4.8 fold greater amounts of CD34+CD45+ HSPCs emerged in both EP- and ST-derived iPS sacs, compared to FB-derived iPS sacs (p<0.01). After an additional 2 weeks of erythroid differentiation, we observed 4.5-8.7 fold greater amounts of GPA+ erythroid cells from both EP- and SC-derived iPS sacs, compared to FB-derived iPS sacs (p<0.01). Interestingly, ST-derived iPS sacs resulted in 1.4-2.0 fold greater amounts of CD34+CD45+ HSPCs and GPA+ erythroid cells (p<0.01), compared to EP-derived iPS sacs. Higher β-globin expression (21.5±4.3%) was observed by RT-qPCR in erythroid cells from ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (4.4±2.5% and 8.3±4.2%, respectively, p<0.01), which was comparable to ES sacs (23.3%). Sickle hemoglobin was detected by hemoglobin electrophoresis. The ES/iPS sac-derived erythroid cell generation was more strongly affected by cell sources (5-6 fold larger SD) than variations among iPS cell clones. These data demonstrate that ST-derived iPS sacs allow more efficient erythroid cell generation with higher β-globin production, compared to EP- and FB-derived iPS sacs. We hypothesized that ST-derived iPS sacs contain greater amounts of immature HSPCs (including hemogenic endothelium) and immature EPs (including megakaryoerythroid progenitors), since more expansion of ST-derived cells was observed during the late phase of erythroid differentiation, compared to EP- and FB-derived cells. We evaluated hemogenic endothelium markers at day 15, and observed 7.7 fold greater amounts of VEGFR+GPA- cells (p<0.01) and 1.3-1.4 fold greater amounts of CD31+CD34+ cells in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (not detectable VEGFR+GPA- cells in EP-derived iPS sacs). Before erythroid differentiation, 3.2-16.4 fold greater amounts of GPA+CD41a+ megakaryoerythroid progenitors were observed in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (p<0.05). In colony forming unit assays, 1.8-5.0 fold greater amounts of myeloid and erythroid colonies were observed in ST-derived iPS sacs, compared to EP- and FB-derived iPS sacs (p<0.01). These data suggest that ST-derived iPS sacs more efficiently produce immature HSPCs and immature EPs, which may result in more efficient generation of erythroid cells with β-globin production. In summary, we demonstrated that human ST-derived iPS sacs allow for more efficient erythroid cell generation with higher β-globin production, which could be caused by heightened emergence of hemogenic endothelium in ST-derived iPS sacs. Our findings should be important for in in vitro iPS cell-derived erythroid cell generation with high β-globin expression. Disclosures Winkler: Novartis: Research Funding; GSK: Research Funding.

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β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs

Blood ◽

10.1182/blood.v124.21.2674.2674 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2674-2674

Author(s):

Naoya Uchida ◽

Atsushi Fujita ◽

Thomas Winkler ◽

John F. Tisdale

Keyword(s):

Progenitor Cells ◽

Erythroid Differentiation ◽

Ips Cells ◽

Cell Fraction ◽

Erythroid Cells ◽

Erythroid Progenitor ◽

Ips Cell ◽

Erythroid Progenitor Cells ◽

Spherical Cells ◽

Induced Pluripotent

Abstract Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell (RBC) transfusion. When ES cell-derived erythroid cells are generated using embryoid bodies, these cells predominantly express embryonic type ε-globin, with lesser fetal type γ-globin and small amounts of adult type β-globin; however, no β-globin expression is detected in iPS cell-derived erythroid cells. Recently, the ES cell-derived sac (ES sac) was reported to express hemangioblast markers and could generate functional platelets (Takayama, Blood. 2008). We previously demonstrated that erythroid cells were also efficiently generated via the ES sac (2013 ASH). We extend this work to evaluate globin expression in ES sac-derived erythroid cells. We generated ES sacs from human H1 ES or iPS cells using VEGF for 15 days, as previously described. The spherical cells within ES sacs were harvested and cultured on OP9 feeder cells for 2 days, and the suspension cells were differentiated into erythroid cells using human erythroid massive amplification culture for 13 days (Blood cells Mol Dis. 2002). The globin types expressed in erythroid cells were evaluated by RT-qPCR and hemoglobin electrophoresis. When hematopoietic cell-stimulating cytokines (SCF, FLT3L, TPO, IL3, EPO, and BMP4) were added in ES sac cultures on day 9-15, we observed 1.4-fold greater amounts of GPA+ erythroid cells (p<0.05) and 1.3-fold lower ε-globin expression in ES sac-derived erythroid cells (p<0.05), suggesting that cytokine stimulation might induce more hematopoietic/stem progenitor cells (HSPC) which can be differentiated to γ- or β-globin-expressing erythroid cells. Thus, we hypothesized that the ES sac contains both primitive and definitive erythroid progenitor cells capable of ε-globin-expression or γ- or β-globin-expression upon differentiation; respectively, and that these progenitors are selectable based upon surface markers of erythroid progenitor cells or HSPCs. To investigate whether primitive erythropoiesis is switched to definitive erythropoiesis during ES sac maturation, we evaluated spherical cells within the ES sac on day 9, 12, 15, and 18 after ES sac culture. A high percentage of GPA+ erythroid cells (29.2±3.7%) were observed on as early as day9. At that time point, almost no CD34+CD45+ HSPCs were present; however, the number increased upon further ES sac maturation until day 15 (6.8±1.6%). Cells further differentiated in erythroid culture had lower ε-globin expression and higher β-globin expression (up to 13.8±1.5%) when harvested from the ES sac at later time points. These data suggest that more matured ES sacs favor less primitive erythropoiesis and more definitive erythropoiesis. On day 15, the ES sacs contained a high percentage of GPA+(CD34-) erythroid cells (68.7±4.0%) and relatively lower amounts of CD34+(GPA-) HSPCs (16.7±2.1%). Therefore, we separated GPA+ and GPA- spherical cells from ES sac by magnetic selection before further erythroid differentiation, which resulted in higher ε-globin expression (43.0±16.6% vs 4.4±1.2%, p<0.01) and lower β-globin expression (7.6±5.3x10e-7% vs 19.8±2.7%, p<0.01) from the GPA+ cell fraction. In contrast, after erythroid differentiation from CD34+ or CD34- sorted spherical cells, lower ε-globin expression (3.7±0.3% vs 17.1±0.9%, p<0.01) and higher β-globin expression (17.4±0.7 % vs 0.9±0.4 %, p<0.01) were observed from the CD34+ cell fraction. These data suggest that the ES sac contains both primitive erythroid progenitor cells in the CD34- or GPA+ cell fraction and definitive erythroid progenitor cells in the CD34+ or GPA- cell fraction. In addition, iPS sac-derived erythroid cells were generated from 2 clones of fibroblast-derived iPS cells, which demonstrated 9.0±2.6% (clone #1) and 7.3±3.7% (clone #2) of β-globin expression. These data demonstrate that similar to ES sac-derived erythroid cells, iPS cell-derived erythroid cells can produce β-globin when differentiated from iPS sacs. In conclusion, we demonstrate that human ES and iPS cells can generate both primitive and definitive erythroid progenitor cells when differentiated in ES/iPS sac. CD34 or GPA discriminates between primitive and definitive erythroid progenitor cells in ES sac. The presented differentiation and selection strategy represent an important step to develop in vitro RBC production system from pluripotent stem cells. Disclosures No relevant conflicts of interest to declare.

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Klf1 Regulatory Networks in Primary Erythroid Cells.

Blood ◽

10.1182/blood.v114.22.1462.1462 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1462-1462

Author(s):

Michael Tallack ◽

Thomas Whitington ◽

Brooke Gardiner ◽

Eleanor Wainwright ◽

Janelle Keys ◽

...

Keyword(s):

Regulatory Networks ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Es Cells ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Gene Promoters ◽

Erythroid Cells ◽

Red Cell Membrane ◽

Sequencing Platform

Abstract Abstract 1462 Poster Board I-485 Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types. Disclosures No relevant conflicts of interest to declare.

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Lack of GDF11 Does Not Ameliorate Erythropoiesis in β-Thalassemia and Does Not Prevent the Activity of the Trap-Ligand RAP-536

Blood ◽

10.1182/blood-2018-165 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 165-165 ◽

Cited By ~ 1

Author(s):

Amaliris Guerra ◽

Rea Oikonomidou ◽

Sinha Gonzalez ◽

Jianbing Zhang ◽

Vania Lo Presti ◽

...

Keyword(s):

Research Funding ◽

Hematological Parameters ◽

Genomic Medicine ◽

Erythroid Differentiation ◽

The United States ◽

Phase Iii ◽

Erythroid Cell ◽

Published Data ◽

Erythroid Cells ◽

Cd34 Cells

Abstract Mutations in the HBB gene causes β-thalassemia (BT). Treatment for BT presents a major clinical challenge in the United States, as patients require chronic and expensive treatment for survival. A new drug in Phase III clinical trials, Luspatercept (ACE-536), has been shown to improve BT symptoms via an erythropoietin (EPO) -independent pathway. ACE-536 is a peptide drug identical to the extracellular domain of activin receptor IIB (ACVR2B). Upon administration, it competes with ACVR2B to bind members of the transforming growth factor (TGF) β superfamily. Growth differentiation factor 11 (GDF11) has been pinpointed as the primary target by which the trap ligand exerts its therapeutic efforts. Studies in murine models of BT using RAP-536 (the mouse analog of ACE-536), have suggested that Gdf11 is overexpressed in erythroblasts and that overexpression functions to inhibit erythroid differentiation. Interestingly, however, ACE-536 and RAP-536 have been shown to stimulate RBC synthesis in healthy humans and mice, where GDF11/Gdf11 overexpression has not been reported. Additionally, the expression data in mice has been questioned because of the unavailability of antibodies that can discriminate between Gdf11 and other TGF-β ligands. Due to the novelty of RAP-536 promoting erythropoiesis through an Epo-independent pathway and the lack of specific antibodies to distinguish between TGF-β ligands, we resorted to genetic tools to investigate the role of Gdf11 in erythropoiesis. For our study, we generated Hbb+/+Gdf11flox/floxand Hbbth3/+Gdf11flox/floxmice and crossed them with EpoRCre and VavCre transgenic lines, resulting in offspring harboring the Gdf11 deletion in erythroid cells and the complete hematopoietic compartment. If Gdf11 is secreted by erythroid cells, and it plays a role in inhibiting erythroid differentiation, then mice lacking Gdf11 in either erythroid cells or all hematopoietic cell lineages should show some increase in red blood cell (RBC) production, hemoglobin (Hb) and hematocrit (Hb). Furthermore, in Hbbth3/+mice, where Gdf11 has been proposed to be overexpressed, improvements in erythroid cell differentiation should be most apparent. Surprisingly, we did not detect any differences in RBC number, Hb or Hct levels of Gdf11 deficient Hbb+/+or Hbbth3/+mice compared to their Gdf11 containing controls. The discrepancy between our results and published data could be explained if Gdf11 is produced by non-hematopoietic tissues and indirectly influences erythropoiesis. Since Gdf11-/-are embryonic-lethal, we crossed Hbb+/+ Gdf11flox/floxand Hbbth3/+ Gdf11flox/flox mice with a tamoxifen (TAM) inducible Cre recombinase under the global Rosa26 promoter (RosaCre) to assess the effect of a pancellular deletion of Gdf11. No detectable differences were found in RBC, Hb or Hct levels of flthese animals after TAM treatment either acutely nor up to 5-6 months post deletion of Gdf11. Administration of RAP-536 significantly improved and increased hematopoietic parameters in the peripheral blood in all six models lacking Gdf11. In the RAP-536-treated Hbbth3/+models, amelioration of anemia was noted by a decrease in spleen size and improved ineffective erythropoiesis indicated by an increased hematological parameters and increased ratio of mature to immature erythroblasts in spleen analyzed by FACS. Therefore, lack of Gdf11 at the erythroid, hematopoietic and pancellular level did not prevent a response to the drug. Next, we investigated the effects of RAP-536 directly on erythroid cells. Since the drug causes increases in RBC and Hb of normal patients, we challenged CD34+ cells with RAP-536 at various concentrations. Results showed no increases in cell numbers, erythroid viability, hemoglobin content nor differentiation. Currently we are investigating the mRNA expression of activin receptors IIA and IIb along with TGF-β ligands in healthy and BT CD34+ cells as well as in erythroid specific progenitors of the Hbbth3/+ mouse model. Our findings suggest that Gdf11 is not the sole target of RAP-536, nor that Gdf11 is required to promote improvement of erythropoiesis. Most importantly, we show that in the absence of Gdf11, RAP-536 is effective at increasing hematological parameters in both Hbb+/+ and Hbbth3/+ mice. The results of this study demonstrate potential alternative target(s) for the action of RAP-536. Future work will focus on identifying the unknown targets of RAP-536. Disclosures Casu: Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Martinez:Acceleron Pharma: Employment. Suragani:Acceleron Pharma: Employment. Kumar:Acceleron Pharma: Employment. Rivella:Ionis Pharmaceuticals, Inc: Consultancy; Protagonist: Consultancy; Disc Medicine: Consultancy; MeiraGTx: Other: SAB.

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LSD1 Plays an Important Role in GATA Switch during Erythroid Differentiation

Blood ◽

10.1182/blood.v122.21.4846.4846 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4846-4846

Author(s):

Yue Jin ◽

Yidi Guo ◽

Dongxue Liang ◽

Yue Li ◽

Zhe Li ◽

...

Keyword(s):

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Es Cells ◽

Regulatory Element ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Mouse Es Cells ◽

Murine Erythroleukemia

Abstract GATA factors play important role in hematopoiesis. In particular, GATA2 is critical for maintenance of hematopoietic stem and progenitor cells (HS/PCs) and GATA1 is required for erythropoiesis. GATA1 and GATA2 are expressed in reciprocal patterns during erythroid differentiation. It was shown that GATA1 occupied the -2.8Kb regulatory element and mediated repression of the GATA2 promoter in terminally differentiating erythroid cells. However, the detailed molecular mechanisms that control the enhancer/promoter activities of the GATA2 gene remain to be elucidated. In this report, we found that LSD1 and TAL1 co-localize at GATA2 1S promoter through ChIP and double-ChIP assays in murine erythroleukemia (MEL) cells. To further test whether LSD1 and its mediated H3K4 demethylation is important for repression of the GATA2 gene during erythroid differentiation, we silenced LSD1 expression in both MEL cells and mouse ES cells using retrovirus mediated shRNA knockdown and induced them to differentiate into erythroid cells with DMSO and EPO, respectively. GATA2 expression was elevated while the level of GATA1 was repressed by RT-qPCR. Furthermore, consistent with the GATA witch hypothesis, ChIP analysis revealed that the levels of H3K4me2 were increased at the GATA2 1S promoter. In addition, knock-down of LSD1 in MEL cells results in inhibition of erythroid cell differenciation and attenuation of MEL cell proliferation and survival. Thus, our data reveal that LSD1 involved in control of terminal erythroid differentiation by regulating GATA switch. The LSD1 histone demethylase complex may be recruited to the GATA2 1S promoter by interacting with TAL1. The H3K4 demethylation activity of LSD1 leads to downregulation of the active H3K4m2 mark at the GATA2 promoter that alters chromatin structure and represses transcription of the GATA2 genes. Disclosures: No relevant conflicts of interest to declare.

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Generation of Induced Pluripotent Stem Cell-Derived Erythroblasts from a Patient with X-Linked Sideroblastic Anemia

Blood ◽

10.1182/blood.v128.22.76.76 ◽

2016 ◽

Vol 128 (22) ◽

pp. 76-76

Author(s):

Shunsuke Hatta ◽

Tohru Fujiwara ◽

Takako Yamamoto ◽

Mayumi Kamata ◽

Yoshiko Tamai ◽

...

Keyword(s):

Bone Marrow ◽

Erythroid Differentiation ◽

Ips Cells ◽

Therapeutic Strategies ◽

Erythroid Cells ◽

Sideroblastic Anemia ◽

Ring Sideroblasts ◽

Induced Pluripotent ◽

Novel Therapeutic Strategies ◽

Novel Therapeutic

Abstract Congenital sideroblastic anemia (CSA) is an inherited microcytic anemia characterized by the presence of bone marrow ring sideroblasts, reflecting excess mitochondrial iron deposition. The most common form of CSA is X-linked sideroblastic anemia (XLSA), which is attributed to mutations in the X-linked gene erythroid-specific 5-aminolevulinate synthase (ALAS2). ALAS2 encodes the enzyme that catalyzes the first and rate-limiting steps in the heme biosynthesis pathway in erythroid cells. This pathway converts glycine and acetyl-coenzyme A to 5-aminolevulinic acid and also requires pyridoxal 5'-phosphate (PLP) as a cofactor. Although PLP has been used for treating XLSA, a marked proportion of patients with XLSA remain refractory to treatment (Ohba et al. Ann Hematol 2013). Therefore, to elucidate the details of the underlying molecular mechanisms that contribute to ringed sideroblast formation as well as to explore novel therapeutic strategies for XLSA, we generated induced pluripotent stem (iPS) cells from a patient with XLSA. Bone-marrow derived mesenchymal stem cells (BM-MSCs) were generated from a healthy volunteer and from the patient with XLSA, who harbored mutations in ALAS2 (c.T1737C, p.V562A). To establish iPS cells, episomal vectors encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, SHP53, and GLIS1 (gift from K. Okita, Kyoto University, Japan) were electroporated into BM-MSCs.The iPS cells were expanded in hESC medium containing DMEM/F-12 and 20% KSR (KnockoutTM Serum Replacement) (Life Technologies). We established one iPS clone from a healthy subject (NiPS) and two clones from the patient with XLSA (XiPS1 and XiPS2). G-band karyotype analysis demonstrated that all three clones had a normal karyotype. Immunocytochemical staining of the clones revealed the expression of transcription factors such as OCT3/4 and NANOG as well as surface markers such as SSEA-4 and TRA-1-60. Pluripotency of each clone was confirmed by the spontaneous differentiation of embryoid bodiesin vitro and teratoma formation in vivo. No clear characteristic differences were observed between XiPS and NiPS. Next, we evaluated the phenotype of iPS-derived erythroid precursors. The iPS cells were induced to undergo erythroid differentiation with Stemline II serum-free medium (Sigma). Both NiPS- and XiPS-derived erythroblasts were nucleated, and predominately expressed embryonic globin genes. Expression profiling of CD235a-positive erythroblasts from NiPS, XiPS1, and XiPS2, revealed 315 and 359 genes that were upregulated and downregulated (>1.5-fold), respectively, in XiPS relative to NiPS. The downregulated genes included globins (HBQ, HBG, HBE, HBD, and HBM) and genes involved in erythroid differentiation (GATA-1, ALAS2, KLF1, TAL1, and NFE2). Gene ontology analysis revealed significant (p < 0.01) enrichment of genes associated with erythroid differentiation, cellular iron homeostasis, and heme biosynthetic processes, implying that heme biosynthesis and erythroid differentiation are compromised in XiPS-derived erythroblasts. Finally, to examine whether XiPS-derived erythroblasts exhibited a phenotype reflective of defective ALAS2 enzymatic activity, we merged the microarray results with a previously reported microarray analysis in which ALAS2 was transiently knocked down using iPS-derived erythroid progenitor (HiDEP) cells (Fujiwara et al. BBRC 2014). The analysis revealed a relatively high degree of overlap regarding downregulated genes in XiPS relative to NiPS, demonstrating a >1.5-fold upregulation and downregulation of eight and 41 genes, respectively. Commonly downregulated genes included those encoding various globins (HBM, HBQ, HBE, HBG, and HBD) and ferritin (FTH1), GLRX5, ERAF, and ALAS2, which are involved in iron/heme metabolism in erythroid cells, suggesting that the phenotype of XiPS-derived erythroid cells resembles that of ALAS2-knockdown HiDEP cells. Interestingly, when the XiPS was induced to undergo erythroid differentiation by co-culture with OP9 stromal cells (ATCC), aberrant mitochondrial iron deposition was detected by prussian blue staining and electron microscope analysis. We are currently conducting biological analyses to characterize established ring sideroblasts. In summary, XiPS can be used as an important tool for clarifying the molecular etiology of XLSA and to explore novel therapeutic strategies. Disclosures Fujiwara: Chugai Pharmaceuticals. Co., Ltd.: Research Funding.

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Expression of ferrochelatase mRNA in erythroid and non-erythroid cells

Biochemical Journal ◽

10.1042/bj2920343 ◽

1993 ◽

Vol 292 (2) ◽

pp. 343-349 ◽

Cited By ~ 25

Author(s):

R Y Y Chan ◽

H M Schulman ◽

P Ponka

Keyword(s):

Sequence Similarity ◽

Erythroid Differentiation ◽

Polyadenylation Signal ◽

Erythroid Cell ◽

Erythroid Cells ◽

Erythroleukemia Cells ◽

Haem Biosynthesis ◽

Polyadenylation Sites ◽

Dna Fragment ◽

Polyadenylation Signals

Ferrochelatase, which catalyses the last step in haem biosynthesis, i.e. the insertion of Fe(II) into protophorphyrin IX, is present in all cells, but is particularly abundant in erythroid cells during haemoglobinization. Using mouse ferrochelatase cDNA as a probe two ferrochelatase transcripts, having lengths of 2.9 kb and 2.2 kb, were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in the non-erythroid tissues and the 2.2 kb transcript more predominant in spleen. In mouse erythroleukemia cells the 2.9 kb ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by dimethyl sulphoxide there is a preferential increase in the 2.2 kb transcript, which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript. Since there is probably only one mouse ferrochelatase gene, the occurrence of two ferrochelatase transcripts could arise from the use of two putative polyadenylation signals in the 3′ region of ferrochelatase DNA. This possibility was explored by using a 389 bp DNA fragment produced by PCR with synthetic oligoprimers having sequence similarity with a region between the polyadenylation sites. This fragment hybridized only to the 2.9 kb ferrochelatase transcript, indicating that the two transcripts differ at their 3′ ends and suggesting that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal. The preferential utilization of the upstream polyadenylation signal may be an erythroid-specific characteristic of ferrochelatase gene expression.

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Abstract 340: From Stem Cells to Cardiomyocytes: HDAC1 Induces Cardiovascular Differentiation by Regulating SOX17-BMP2 Expression in Early Development.

Circulation Research ◽

10.1161/res.111.suppl_1.a340 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Eneda Hoxha ◽

Erin Lambers ◽

Veronica Ramirez ◽

Prasanna Krishnamurthy ◽

Suresh Verma ◽

...

Keyword(s):

Stem Cells ◽

Molecular Mechanisms ◽

Es Cells ◽

Ips Cells ◽

Full Potential ◽

Pluripotent Cells ◽

Differentiation Potential ◽

Histone Deacetylase 1 ◽

Specific Differentiation ◽

Induced Pluripotent

Cardiomyocytes derived from embryonic and induced pluripotent stem cells (ES/iPS) provide an excellent source for cell replacement therapies following myocardial ischemia. However, some of the obstacles in the realization of the full potential of iPS/ES cells arise from incomplete and poorly understood molecular mechanisms and epigenetic modifications that govern their cardiovascular specific differentiation. We identified Histone Deacetylase 1 (HDAC1) as a crucial regulator in early differentiation of mES and iPS cells. We propose a novel pathway in which HDAC1 regulates cardiovascular differentiation by regulating SOX17 which in turn regulates BMP2 signaling in differentiating pluripotent cells. Utilizing stable HDAC1 knock-down (HDAC1-KD) cell lines, we report an essential role for HDAC1 in deacetylating regulatory regions of pluripotency-associated genes during early cardiovascular differentiation. HDAC1-KD cells show severely repressed cardiomyocyte differentiation potential. We propose a novel HDAC1-BMP2-SOX17 dependent pathway through which deacetylation of pluripotency associated genes leads to their suppression and allows for early cardiovascular-associated genes to be expressed and differentiation to occur. Furthermore, we show that HDAC1 affects DNA methylation both during pluripotency and differentiation and plays a crucial, non-redundant role in cardiovascular specific differentiation and cardiomyocyte maturation. Our data elucidates important differences between ES and iPS HDAC1-KD cells that affect their ability to differentiate into cardiovascular lineages. As varying levels of chromatin modifying enzymes are likely to exist in patient derived iPS cells, understanding the molecular circuitry of these enzymes in ES and iPS cells is critical for their potential therapeutic applications in regenerative medicine. Further research in the molecular mechanisms involved in this process will greatly aid our understanding of the epigenetic circuitry of pluripotency and differentiation in pluripotent cells.

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Heme-Regulated Inhibitor (HRI) Activates Transcription Factor ATF4 to Promote BCL11A Transcription and Fetal Hemoglobin Silencing

Blood ◽

10.1182/blood-2019-123837 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 814-814

Author(s):

Peng Huang ◽

Scott A. Peslak ◽

Xianjiang Lan ◽

Eugene Khandros ◽

Malini Sharma ◽

...

Keyword(s):

Transcription Factor ◽

Cell Line ◽

Research Funding ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Mrna Levels ◽

Globin Genes ◽

Erythroid Cells ◽

Major Pathway ◽

Specific Regulation

Reactivation of fetal hemoglobin in adult red blood cells benefits patients with sickle cell disease and β-thalassemia. BCL11A is one of the predominant repressors of fetal γ-globin transcription and stands as an appealing target for therapeutic genome manipulation. However, pharmacologic perturbation of BCL11A function or its co-regulators remains an unmet challenge. Previously, we reported the discovery of the erythroid-enriched protein kinase HRI as a novel regulator of γ-globin transcription and found that HRI functions in large part via controlling the levels of BCL11A transcription (Grevet et al., Science, 2018). However, the specific mechanisms underlying HRI-mediated modulation of BCL11A levels remain unknown. To identify potential HRI-controlled transcription factors that regulate BCL11A, we performed a domain-focused CRISPR screen that targeted the DNA binding domains of 1,447 genes in the human erythroid cell line HUDEP2. Activating transcription factor 4 (ATF4) emerged as a novel γ-globin repressor. Prior studies reported that ATF4 production is under positive influence of HRI. Specifically, HRI phosphorylates translation factor EIF2α which in turn augments translation of ATF4 mRNA. As expected, HRI deficiency reduced ATF4 protein amounts in HUDEP2 and primary erythroid cells. We further found that the degree of γ-globin reactivation was similar in ATF4 and HRI-depleted cells. ATF4 ChIP-seq in both HUDEP2 and primary erythroblast identified 4,547 and 3,614 high confidence binding sites, respectively. Notably, we did not observe significant enrichment of ATF4 binding or even the presence of an ATF4 consensus motif at the γ-globin promoters, suggesting that ATF4 regulates the γ-globin genes indirectly. However, ATF4 specifically bound to one of the three major BCL11A erythroid enhancers (+55) in both cell types. This was the sole binding site within the ~0.5Mb topologically associating domain that contains the BCL11A gene. Eliminating this ATF4 motif via CRISPR guided genome editing lowered BCL11A mRNA levels and increased γ-globin transcription. Capture-C showed that ATF4 knock-out or removal of the ATF4 site at the BCL11A (+55) enhancer decreased chromatin contacts with the BCL11A promoter. Forced expression of BCL11A largely restored γ-globin silencing in cells deficient for ATF4 or lacking the ATF4 motif in the BCL11A (+55) enhancer. An unexplained observation from our prior study was that HRI loss did not significantly lower Bcl11a levels in murine erythroid cells. Therefore, we mutated the analogous ATF4 motif in the Bcl11a enhancer in the murine erythroid cell line G1E. Unlike in human cells, Bcl11a mRNA synthesis was decreased only very modestly, and there was no effect on the murine embryonic globin genes whose silencing requires Bcl11a. This suggests that the species specific regulation of BCL11A by HRI results from divergent functional roles of ATF4 binding at the BCL11A (+55) enhancer. In sum, our studies uncover a major pathway that extends linearly from HRI to ATF4 to BCL11A to γ-globin. Moreover, these results further support HRI as a pharmacologic target for the selective regulation of BCL11A and γ-globin. Disclosures Blobel: Pfizer: Research Funding; Bioverativ: Research Funding.

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Analysis of Viral Integration Sites in Human Induced Pluripotent Stem Cells.

Blood ◽

10.1182/blood.v114.22.1485.1485 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1485-1485

Author(s):

Thomas Winkler ◽

Amy R Cantelina ◽

Jean-Yves Metais ◽

Xiuli Xu ◽

Anh-Dao Nguyen ◽

...

Keyword(s):

Transcription Factors ◽

Gene Transfer ◽

Cell Lines ◽

Chromosomal Location ◽

Somatic Cells ◽

Ips Cells ◽

Ips Cell ◽

Integration Sites ◽

Equity Ownership ◽

Induced Pluripotent

Abstract Abstract 1485 Poster Board I-508 The recently discovered approach for the direct reprogramming of somatic cells into induced pluripotent stem (IPS) cells by expression of defined transcription factors may provide new approaches for regenerative medicine, gene therapy and drug screening. Successful reprogramming currently requires at least temporary expression of one to four different transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog and Lin28) in the targeted cells. Non-viral based reprogramming technologies have been reported, but expression of the reprogramming factors after γ-retroviral or lentiviral gene transfer remains the most efficient and commonly used approach. Since the reprogramming frequency is consistently low in these studies, it has been speculated that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. Here we present for the first time an extensive analysis of the lentiviral integration profile in human IPS-cells. We analysed the IS of 8 IPS cell lines derived from either human fetal fibroblasts (IMR90) or newborn foreskin fibroblasts (FS) after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28, using linear amplification-mediated PCR (LAM-PCR). With 5 to15 IS per individual IPS clone we identified a total of 78 independent IS. Finally we assigned 75 IS to a unique chromosomal location. In addition to LAM-PCR, we confirmed the total number of IS via Southern blot. Interestingly, in 6 of 8 IPS clones some of these IS were found in pairs, integrated into the same chromosomal location within 4 base pairs of each other. This integration pattern has not been detected in our previous analysis of 702 IS in rhesus macaques transplanted with CD34+ cells transduced with retroviral vectors. Of the 75 valid IS 53 (70.7%) could be mapped to a gene-coding region, 52 located in introns and 1 in an exon, annotated in a human reference sequence in the UCSC Genome Browser RefSeq Genes track. The different IPS-clones had no integration site in common. To investigate the impact of integration on the regulation of vector targeted genes we analyzed the mRNA expression profiles using available microarray data from these clones. Out of 46 evaluable genes only two (WDR66 and MYST2 in clone IMR90-2, p<0.0001) were significantly over-expressed. The expression of two genes in clone FS-1 (ACVR2A p=0.01, RAF1 p=0.02) and one in FS-2 (KIAA0528, p=0.03) was decreased compared to the expression data of all other clones combined. In summary our data suggest that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes. Furthermore, identification of the insertion profile of the IPS cell clones IMR90-1 and -4 as well as FS-1 will be useful to other researchers using these cell lines distributed by the Wisconsin International Stem Cell (WISC) bank. Disclosures: Antosiewicz-Bourget: Cellular Dynamics International: Consultancy, Equity Ownership. Thomson: Cellular Dynamics International: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Dunbar: ASH: Honoraria.

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Studies on Heme Oxygenase 1 During Erythroid Differentiation

Blood ◽

10.1182/blood.v116.21.4254.4254 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4254-4254

Author(s):

Daniel Garcia Santos ◽

Jesse Eisenberg ◽

Matthias Schranzhofer ◽

Prem Ponka

Keyword(s):

Aminolevulinic Acid ◽

Conflicts Of Interest ◽

Tissue Injury ◽

Erythroid Differentiation ◽

The Body ◽

Cellular Damage ◽

Erythroid Cell ◽

Heme Oxygenase 1 ◽

Erythroid Cells ◽

Murine Erythroleukemia

Abstract Abstract 4254 Heme is indispensable for the function of all aerobic cells as a prosthetic group of innumerable proteins. However, “free heme” (uncommitted) can initiate the formation of free radicals and cause lipid peroxidation, which can lead to cellular damage and tissue injury. Therefore, the rate of heme biosynthesis and catabolism must be well balanced by tight control mechanisms. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC), whose precursors synthesize heme with rates that are at least one order of magnitude higher (on the per cell basis) than those in the liver – the second most active heme producer in the body. The degradation of heme is exclusively carried out by heme oxygenases 1 and 2 (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using both a murine erythroleukemia cell line (MEL) and primary erythroid cells isolated from mouse fetal livers, we have demonstrated that during erythroid differentiation HO1 is up-regulated at both mRNA and protein levels. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase. These data suggest that in developing RBC, in addition to the continuous assembly of heme with globin chains, there is an increase in levels of uncommitted heme, which upregulates HO1 expression. Additionally, we have shown that down-regulation of HO1 via siRNA increased hemoglobinization in differentiating MEL cells. In contrast, induction of HO1 expression by NaAsO2 reduced the hemoglobinization of MEL cells. This effect could be reversed to control levels by the addition of HO1 inhibitor tin-protophorphyrin (SnPP). These results show that in differentiating erythroid cells the balance between levels of heme and HO1 have to be tightly regulated to maintain hemoglobinization at appropriate levels. Our results lead us to propose that disturbances in HO1 expression could play a role in some pathophysiological conditions such as thalassemias. Disclosures: No relevant conflicts of interest to declare.

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