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

Blood ◽  
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.

scholarly journals Generation of Patient-Derived Human Induced Pluripotent Stem Cells in X-Linked Sideroblastic Anemia and Novel Therapeutic Strategies

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 938-938
Author(s):  
Yuki Morimoto ◽  
Kazuhisa Chonabayashi ◽  
Masayuki Umeda ◽  
Hiroshi Kawabata ◽  
Akifumi Takaori-Kondo ◽  
...  
Keyword(s):  
X Chromosome ◽  
Pluripotent Stem Cells ◽  
Molecular Mechanisms ◽  
Aminolevulinic Acid ◽  
Ips Cells ◽  
Erythroid Cells ◽  
Wild Type ◽  
Sideroblastic Anemia ◽  
Induced Pluripotent ◽  
In Erythroid Cells

Abstract Sideroblastic anemias consist of a heterogeneous group of inherited and acquired disorders. The most common hereditary type is X-linked sideroblastic anemia (XLSA), which is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Heme synthesis starts with the polymerization of glycine and succinyl CoA polymerization and synthesis of δ-aminolevulinic acid (ALA) in the mitochondria. ALAS2 encodes the enzyme that catalyzes these first steps in the heme synthetic pathway in erythroid cells, steps that require pyridoxal 5'-phosphate (PLP) as a cofactor. It has been found that treatment with PLP is effective for a small fraction of XLSA patients, but there are no effective treatments for the other fraction. The aim of this study is to explore the molecular mechanisms of XLSA and to develop new effective therapies. We used episomal methods to generate induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) of three late-onset XLSA female patients in one family. The cells harbored the heterozygous mutation (R227C) in the ALAS2 gene. Because ALAS2 is located in the X-chromosome, either wild-type or mutant ALAS2 gene is inactivated in the erythroid cells of female heterozygotes. All three patients showed severe anemia and their PBMCs showed skewed X-chromosome inactivation with preferential inactivation of the X chromosome carrying wild-type ALAS2, indicating a condition associated with unbalanced lyonization. From each patient, we successfully established iPSC lines with the active mutant ALAS2 allele and with the active wild-type ALAS2 allele. We assessed the hematopoietic differentiation potential of these two types of iPSC lines derived from the same patient. Differentiation into hematopoietic progenitor cells (HPCs) using embryoid body formation was comparable in the two groups. However, further differentiation in erythroid culture was significantly impaired in iPSC lines harboring the active mutant ALAS2 allele compared with those harboring the active wild-type ALAS2 allele (CD235a+ cells: 59.20±12.16% with the active wild-type ALAS2 allele vs. 3.95±4.71% with the active mutant ALAS2 allele, p&lt;0.01). Only mutant ALAS2 expression was observed in erythroid cells differentiated from iPS cells harboring the active mutant ALAS2 allele, and only wild-type ALAS2 expression was observed in erythroid cells differentiated from iPS cells harboring the active wild-type ALAS2 allele. Hematopoietic maturation capacity was assessed by performing colony-forming unit (CFU) assays of HPCs (CD34+CD38-CD43+lineage marker-) from iPSC lines derived from the same XLSA patient. Erythroid colony count was significantly less in HPCs from iPSC lines with the active mutant ALAS2 allele, but there was no difference in total colony count between the two types of iPSC lines (erythroid colony numbers: 9.66±10.69 vs. 0±0 per 7,500 HPCs, p&lt;0.01; mixed erythroid colony numbers: 15.00±11.26 vs. 0.66±0.57 per 7,500 HPCs, p&lt;0.01; HPCs with the active wild-type ALAS2 allele vs. HPCs with the active mutant ALAS2 allele). We examined the effect of ALA on the erythroid differentiation of the HPCs. The CD235a-positive erythroid cell ratio of HPCs with the active wild-type ALAS2 allele did not increase following administration of ALA. By contrast, the ratio reached normal levels for HPCs with the active mutant ALAS2 allele (CD235a+ cells: 6.10± 5.61% vs. 85.34± 11.05%, p&lt;0.01; without vs. with administration of ALA). Our data suggest that our iPSC-based system could be useful for studying the precise molecular mechanisms of XLSA and drug testing. Figure Figure. Disclosures Morimoto: Grant-in-Aid for JSPS Research Fellow: Research Funding. Takaori-Kondo: celgene: Honoraria, Research Funding; Bristol-Myers Squibb, Novartis, Janssen pharma, Pfizer: Honoraria.

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

Blood ◽  
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.

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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1150-1150
Author(s):  
Naoya Uchida ◽  
Fujita Atsushi ◽  
Haro-Mora J Juan ◽  
Thomas Winkler ◽  
John F Tisdale
Keyword(s):  
Research Funding ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Ips Cells ◽  
Cell Generation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Hemogenic Endothelium ◽  
Ips Cell ◽  
Induced Pluripotent

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.

scholarly journals Generation and Molecular Characterization of Human Ring Sideroblasts: a Key Role of Ferrous Iron in Terminal Erythroid Differentiation and Ring Sideroblast Formation

2019 ◽  
Vol 39 (7) ◽  
Author(s):  
Kei Saito ◽  
Tohru Fujiwara ◽  
Shunsuke Hatta ◽  
Masanobu Morita ◽  
Koya Ono ◽  
...  
Keyword(s):  
Ferrous Iron ◽  
Induced Pluripotent Stem Cell ◽  
Erythroid Differentiation ◽  
Binding Motif ◽  
Sideroblastic Anemia ◽  
Metal Transporters ◽  
Ferrous Citrate ◽  
Ring Sideroblasts ◽  
Undifferentiated Cells ◽  
Induced Pluripotent

ABSTRACT Ring sideroblasts are a hallmark of sideroblastic anemia, although little is known about their characteristics. Here, we first generated mutant mice by disrupting the GATA-1 binding motif at the intron 1 enhancer of the ALAS2 gene, a gene responsible for X-linked sideroblastic anemia (XLSA). Although heterozygous female mice showed an anemic phenotype, ring sideroblasts were not observed in their bone marrow. We next established human induced pluripotent stem cell-derived proerythroblast clones harboring the same ALAS2 gene mutation. Through coculture with sodium ferrous citrate, mutant clones differentiated into mature erythroblasts and became ring sideroblasts with upregulation of metal transporters (MFRN1, ZIP8, and DMT1), suggesting a key role for ferrous iron in erythroid differentiation. Interestingly, holo-transferrin (holo-Tf) did not induce erythroid differentiation as well as ring sideroblast formation, and mutant cells underwent apoptosis. Despite massive iron granule content, ring sideroblasts were less apoptotic than holo-Tf-treated undifferentiated cells. Microarray analysis revealed upregulation of antiapoptotic genes in ring sideroblasts, a profile partly shared with erythroblasts from a patient with XLSA. These results suggest that ring sideroblasts exert a reaction to avoid cell death by activating antiapoptotic programs. Our model may become an important tool to clarify the pathophysiology of sideroblastic anemia.

scholarly journals Generation and Molecular Characterization of Human Ring Sideroblasts

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3613-3613
Author(s):  
Kei Saito ◽  
Tohru Fujiwara ◽  
Shunsuke Hatta ◽  
Chie Suzuki ◽  
Noriko Fukuhara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Molecular Characterization ◽  
Ferrous Iron ◽  
Research Funding ◽  
Erythroid Differentiation ◽  
Wild Type ◽  
Sideroblastic Anemia ◽  
Iron Transporter ◽  
Ring Sideroblasts

Abstract (Background) Sideroblastic anemias are heterogeneous congenital and acquired refractory anemias characterized by bone marrow ring sideroblasts, reflecting excess mitochondrial iron deposition. While the disease is commonly associated with myelodysplastic syndrome, the congenital forms of sideroblastic anemias comprise a diverse class of syndromic and non-syndromic disorders, which are caused by the germline mutation of genes involved in iron-heme metabolism in erythroid cells. Although the only consistent feature of sideroblastic anemia is the bone marrow ring sideroblasts, evidence on the detailed molecular characteristics of ring sideroblasts is scarce owing to a lack of the biological models. We have recently established ring sideroblasts by inducing ALAS2 gene mutation based on human-induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells (ASH 2017) and have further extended the molecular characterization of human ring sideroblasts to gain new biological insights. (Method) We targeted the GATA-1-binding region of intron 1 of the human ALAS2 gene in HiDEP cells and established two independent clones [X-linked sideroblastic anemia (XLSA) clones]. A co-culture with OP9 stromal cells (ATCC) was conducted with IMDM medium supplemented with FBS, erythropoietin, dexamethasone, MTG, insulin-transferrin-selenium, and ascorbic acid. To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in a liquid suspension culture (Fujiwara et al. JBC 2014). Bone marrow glycophorin A (GPA)-positive erythroblasts of patients with XLSA and normal individuals were separated using the MACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) after obtaining written informed consent. For transcription profiling, Human Oligo chip 25K (Toray) was used. (Results) We previously demonstrated that co-culture with OP9 cells in the medium supplemented with 100 uM sodium ferrous citrate (SFC) promoted erythroid differentiation of XLSA clones, which enabled the establishment of ring sideroblasts (ASH 2017). To confirm the importance of SFC in terminal erythroid differentiation, we further demonstrated that the addition of SFC, and not transferrin-loaded iron, induced the frequency of GPA+ cells and TfR1-GPA+ mature erythroid population, based on primary erythroblasts derived from human CD34-positive cells. Subsequently, to reveal the molecular mechanism by which abnormal iron mitochondrial iron accumulation occurs by co-culture with SFC, we evaluated the expressions of various metal transporters, demonstrating that the addition of SFC significantly increased the expressions of mitoferrin 1 (MFRN1; a ferrous iron transporter in mitochondria), divalent metal transporter 1 (DMT1), and Zrt- and Irt-like protein 8 (ZIP8; a transmembrane zinc transporter, recently known as a ferrous iron transporter) in the XLSA clone than the wild-type cells, which would have contributed to the formation of ring sideroblasts. Moreover, we performed expression analyses to elucidate the biochemical characteristics of ring sideroblasts. After co-culture with OP9 in the presence of SFC, ring sideroblasts exhibited more than two-fold upregulation and downregulation of 287 and 143 genes, respectively, than the wild-type cells. Interestingly, when compared with the expression profiling results before co-culture (ASH 2017), we noticed prominent upregulation of gene involved in anti-apoptotic process (p = 0.000772), including HSPA1A, superoxide dismutase (SOD) 1, and SOD2. In addition, we conducted a microarray analysis based on GPA-positive erythroblasts from an XLSA patient and a normal individual. The analysis revealed significant upregulation of genes involved in the apoptosis process, as represented by apoptosis enhancing nuclease, DEAD-box helicase 47, and growth arrest and DNA-damage-inducible 45 alpha, and anti-apoptotic genes, such as HSPA1A and SOD2. Concomitantly, when the XLSA clone was co-cultured with OP9 in the presence of SFC, the apoptotic cell frequency as well as DNA fragmentation were significantly reduced compared with the XLSA clone co-cultured without SFC, indicating that ring sideroblasts avoid cell death by inducing anti-apoptotic properties. (Conclusion) Further characterization of the XLSA model would help clarify its molecular etiology as well as establish novel therapeutic strategies. Disclosures Fukuhara: Celgene: Research Funding; Chugai: Research Funding; Daiichi-Sankyo: Research Funding; Boehringer Ingelheim: Research Funding; Eisai: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Janssen: Honoraria, Research Funding; Japan Blood Products Organization: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Mitsubishi Tanabe: Research Funding; Mundipharma: Honoraria, Research Funding; MSD: Research Funding; Nippon-shinyaku: Research Funding; Novartis pharma: Research Funding; Ono: Honoraria, Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; Sanofi: Research Funding; Symbio: Research Funding; Solasia: Research Funding; Sumitomo Dainippon: Research Funding; Taiho: Research Funding; Teijin Pharma: Research Funding; Zenyaku Kogyo: Honoraria, Research Funding; Takeda: Honoraria; Baxalta: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Bayer Yakuhin: Research Funding; Alexionpharma: Research Funding; AbbVie: Research Funding; Astellas: Research Funding; Nihon Ultmarc: Research Funding.

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