Generation and Molecular Characterization of Human Ring Sideroblasts
Authors: Saito Kei , Fujiwara Tohru , Hatta Shunsuke , Suzuki Chie , Fukuhara Noriko , Onishi Yasushi , Nakamura Yukio , Harigae Hideo 
Published: October 2019

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.