A Defined Culture Method Enabling the Establishment of Ring Sideroblasts

LETTERS TO THE EDITOR

Table 1. Hematologic and biochemical data. A defined culture method enabling the Hematological data Normal range establishment of ring sideroblasts from induced pluripotent cells of X-linked sideroblastic anemia White blood cells 3,800/µL (4,000 - 9,000) Neutrophils 34% (28 - 68) Congenital sideroblastic anemia is an inherited anemia Lymphocytes 57% (17 - 57) characterized by the presence of bone marrow ring sideroblasts.1 To date, several causative genes, mostly related to Monocytes 4% (0 - 10) iron and heme metabolism in erythroid cells, have been Eosinophils 4% (0 - 10) 1 considered critical for the formation of ring sideroblasts. Basophils 1% (0 - 2) However, the biological characteristics of ring sideroblasts Red blood cells 450 x 104/ L (427 - 570 x 104) remain elusive because of the lack of an experimental µ model. We established culture conditions to induce ring Hemoglobin 8.1 g/dL (14 - 18) sideroblasts in vitro based on induced pluripotent stem (iPS) Mean corpuscular volume 57.7 fL (80 - 100) cells derived from a patient with congenital sideroblastic Mean corpuscular hemoglobin 31.1% (31 - 35) anemia. To our knowledge, this is the first successful establishment of ring sideroblasts. concentration X-linked sideroblastic anemia (XLSA), the most common Reticulocyte 2.2% (0.7 - 2) type of congenital sideroblastic anemia, is caused by Platelets 60.1 x 104// L (15 - 35 x 104) defects in the X-linked gene encoding 5-aminolevulinic µ 1,2 acid synthase 2 (ALAS2). Most XLSA-associated muta- Biochemical data Normal range tions in ALAS2 are missense substitutions that cause the loss of protein function, whereas mutations in the ALAS2 Total protein 6.9 g/dL (6.7-8.1) regulatory region, which includes the promoter and intron Albumin 4.3 g/dL (3.8-5.3) 1,1 cause decreased ALAS2 expression. Missense mutations commonly decrease the binding of ALAS2 to pyridox- Aspartate transaminase 14 U/L (8-38) al 5 -phosphate (vitamin B6), a cofactor for the enzymatic Alanine transaminase 8 IU/L (4-43) activity′ of ALAS2, accounting for the responsiveness of Alkaline phosphatase 979 IU/L (115-359) patients with XLSA harboring such mutations to pyridoxal Lactate dehydrogenase 155 IU/L (119-229) 5 -phosphate treatment.1 However, approximately half of XLSA′ cases are unresponsive to pyridoxal 5 -phosphate,1,2 Gamma-glutamyltransferase 14 mg/dL (10-47) necessitating the exploration of novel therapeutic′ strate- Total bilirubin 1.6 mg/dL (0,2-1) gies. Blood urea nitrogen 11 mg/dL (8-20) An in vivo model of XLSA is, therefore, needed to eluci- date the underlying molecular mechanisms that contribute Creatinine 0.5 mg/dL (0.44-1.15) to the formation of ring sideroblasts and explore novel ther- C-reactive protein 0.1 mg/dL (0-0.14) apeutic strategies. However, Alas2-knockout mice die by Iron 242 g/dL (54-181) day 11.5 in utero and are not, therefore, available for further Total iron binding capacity 279 g/dL (231-385) testing.3 Moreover, although ring sideroblast formation is observed after the transgenic rescue of ALAS2 in Alas2- Ferritin 223 ng/mL (50-200) knockout mice, these mice die soon after birth.4 Heme-defi- Abnormal values are shown in bold. cient erythroblasts, derived from Alas2-knockout embryonic stem cells, through in vitro culture with OP9 cells, exhibited aberrant cytoplasmic iron accumulation but failed to present as typical ring sideroblasts.5 These previous find- Supplementary Figure S2A), differentiation potential into adi- ings highlight the need for a model of XLSA. pogenic/osteogenic lineages (Online Supplementary Figure Since the first report on iPS cells, various disease-specific S2B), and immunophenotypes (Online Supplementary Figure human iPS cells have been established to partially recapit- S2C). Additionally, Sanger sequencing confirmed ALAS2 ulate disease phenotypes.6 However, while such iPS cell mutations in these mesenchymal stem cells (Online models have been established for hematologic disorders, Supplementary Figure S2D). Similarly, we established bone no previous reports have described the establishment of an marrow mesenchymal stem cells from a healthy donor. iPS cell model of XLSA. We, therefore, attempted to estab- The bone marrow mesenchymal stem cells derived from lish an iPS cell line from a patient with XLSA. The study a healthy donor and the patient with XLSA were repro- protocol was approved by the Ethics Committee of Tohoku grammed to iPS cells (termed NiPS and XiPS cells, respec- University Graduate School of Medicine. Clinical samples tively) using episomal vectors encoding OCT3/4, NANOG, were collected after obtaining written informed consent. SOX2, KLF4, L-MYC, LIN28, GLIS1, and shp53 (p53 A 14-year old male had exhibited microcytic anemia shRNA) (Online Supplementary Figure S3A). One NiPS cell since childhood (Table 1). Bone marrow analysis revealed clone and one XiPS cell clone were selected for further ring sideroblasts (50% of all erythroblasts) (Online characterization. G-band karyotype analysis confirmed Supplementary Figure S1A) with normal karyotype. The normal karyotypes. Additionally, immunocytochemical genetic analysis identified a thymine-to-cytosine substitu- staining of the iPS cell clones confirmed the expression of tion in exon 11 (T1737C) that caused a change in the transcription factors OCT3/4 and NANOG and surface 562nd amino acid of the ALAS2 polypeptide from valine to markers SSEA-4 and TRA-1-60, which are characteristic of alanine (Val562Ala) (Online Supplementary Figure S1B). The human embryonic stem cells (Online Supplementary Figure patient was diagnosed with XLSA and found to be respon- S3B). Next, we confirmed the pluripotency of both NiPS sive to vitamin B6. We established bone marrow mes- and XiPS cells based on the spontaneous differentiation of enchymal stem cells from the patient’s bone marrow sam- embryoid bodies in vitro (Online Supplementary Figure S4A) ple (Online Supplementary Figure S2). The phenotype of and teratoma formation in vivo (Online Supplementary Figure these cells was confirmed based on morphology (Online S4B). No significant differences were observed between

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XiPS and NiPS cells regarding iPS cell induction efficiency XLSA-iPS cells from bone marrow mesenchymal stem cells or characterization (Online Supplementary Figures S3 and using a non-integrating episomal vector7 encoding seven S4). Furthermore, Sanger sequencing confirmed the pres- factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, LIN28, GLIS1, ence of an ALAS2 mutation in XiPS cells (Figure 1A). and shp53). Transfection efficiency into bone marrow mes- Regarding the combination of reprogramming factors, enchymal stem cells is not satisfactory,8 but our approach introduction of LIN28, shp53, and GLIS1 in addition to the could be applied to various cell types for establishing iPS four originally reported factors (i.e., OCT3/4, SOX2, MYC, cells. and KLF4) may enhance the efficiency of iPS cell establish- Furthermore, co-culture of iPS cells with stromal cell ment.7 Here, we successfully and efficiently established lines has commonly been used to obtain erythroblasts from

Figure 1. Establishment of ring sideroblasts in vitro from XiPS cells. (A) Sanger sequencing of NiPS and XiPS cells showing a T to C substitution (c.1737) in the ALAS2 gene in XiPS cells. (B) Experimental protocol for co-culture with stromal cells. Cells were re-seeded onto mitomycin C-treated stromal cells on day 15; the medium was supplemented on day 21 with sodium ferrous citrate and incubated for an additional 4 days. BMP4, bone morphogenetic protein 4; VEGF, vascular endothelial growth factor; SCF, stem cell factor; IL- 3, interleukin-3; EPO, erythropoietin; TPO, thrombopoietin; SFC, sodium ferrous citrate; IMDM, Iscove modified Dulbecco medium; FBS, fetal bovine serum. (C) Prussian blue staining (top) and electron microscopy (middle and bottom) of NiPS- and XiPS-derived erythroblasts. For Prussian blue staining, typical ring sideroblasts are indicated by arrows. For the lower magnification of electron microscopy, mitochondrial iron deposits are indicated by arrows. For the higher magnification, normal- and iron-loaded mitochondria, derived from NiPS and XiPS, respectively, are indicated.

haematologica 2018; 103:e189 LETTERS TO THE EDITOR

iPS cells.9 We, therefore, differentiated NiPS and XiPS cells thesis (e.g., SLC25A38 and FECH), iron–sulfur cluster bio- into erythroid cells by co-culturing with stromal cells. iPS genesis (e.g., ABCB7, GLRX5, and HSPA9), mitochondrial cells were seeded onto mitomycin C-treated stromal cells protein synthesis (e.g., TRNT1, YARS2, PUS1, SLC19A2, (OP9 or C3H/10T1/2; American Type Culture Collection, NDUFV11, and mitochondrial DNA), and RNA splicing Manassas, VA, USA) with Iscove modified Dulbecco medi- machinery (e.g, SF3B1).1,11-13 Recently, one report on iPS um (IMDM; Life Technologies) supplemented with fetal cells from a patient with a mitochondrial deletion/Pearson bovine serum, insulin–transferrin–selenium, bone morpho- syndrome implicated the formation of ring sideroblasts; genetic protein 4 (40 ng/mL), and vascular endothelial however, this was not confirmed with electron 14 growth factor (20 ng/mL) (Figure 1B). On day 3, this medi- microscopy. No effective treatment has been established um was replaced with fresh IMDM (Sigma-Aldrich) supple- for sideroblastic anemia not related to the ALAS2 mutation, mented with fetal bovine serum, insulin–transferrin–seleni- but our defined culture protocol would lead to better um, and vascular endothelial growth factor (20 ng/mL). On understanding of the pathophysiology of both congenital day 14 or 15, total cells were harvested, filtered (70 m), and acquired sideroblastic anemias. and seeded onto mitomycin C-treated stromal cells μ with To the best of our knowledge, no previous report has IMDM (Sigma-Aldrich) supplemented this time with fetal described the establishment of ring sideroblasts through in bovine serum, insulin–transferrin–selenium, stem cell fac- vitro culture. One potential limitation of the present proto- tor (40 ng/mL), interleukin-3 (10 ng/mL), erythropoietin (3 col is that only a subset of the XiPS cell-derived erythrob- IU/mL), and thrombopoietin (20 ng/mL). On day 20 or 21, lasts (approximately 20%) exhibited ring sideroblasts, a problem which may be overcome by establishing an the cell culture was supplemented with 250 µM sodium ferrous citrate and incubated for an additional 4 days. The immortalized erythroblast clone with ring sideroblasts number of erythroblasts decreased drastically by day 28, so based on viral transduction of the hematopoietic transcription factor TAL1 and human papillomavirus type 16- the cells were harvested by day 25. 15 Interestingly, approximately 20% of erythroblasts E6/E7. Nevertheless, further phenotypic analyses of ring obtained from XiPS cells exhibited ring sideroblasts and sideroblasts could lead to a better understanding of the abnormal iron deposition in the mitochondria, as con- molecular basis of XLSA and the establishment of novel firmed by electron microscopy (Figure 1C); without the therapeutic strategies. addition of sodium ferrous citrate, obvious ring sideroblast Shunsuke Hatta,1,2 Tohru Fujiwara,1 Takako Yamamoto,2 formation was not confirmed. Prolonged exposure (>5 1 1 3 days) or higher concentrations of exogenous iron (>250 Kei Saito, Mayumi Kamata, Yoshiko Tamai, Shin Kawamata2 and Hideo Harigae1 µM) did not increase the proportion of ring sideroblasts (data not shown). On the other hand, abnormal iron deposi- 1Department of Hematology and Rheumatology, Tohoku University tion was not detected in NiPS cell-derived erythroblasts Graduate School of Medicine, Sendai; 2Division of Cell Therapy, (Figure 1C). May-Giemsa staining of erythroblasts derived Foundation of Biomedical Research and Innovation, Kobe and from XiPS and NiPS cells indicated orthochromatic stages 3Department of Gastroenterology and Hematology, Hirosaki University of erythroblasts (Online Supplementary Figure S5), suggest- Graduate School of Medicine, Japan ing that erythroid differentiation was not compromised Acknowledgments: we thank Dr. Ryo Ichinohasama (Tohoku despite the ALAS2 mutation in this in vitro system. In con- University) for helpful comments regarding the histological examination junction with the observation that ferritin clumps were and Ms. Kumiko An (Tohoku University Hospital) for conducting present only in XiPS cell-derived erythroblasts (Figure 1C, the G-band analysis. We also thank Dr. Okita (Center for iPS Cell lower magnification), we anticipate that defective iron uti- Research and Application, Kyoto University, Kyoto, Japan) for kindly lization in ALAS2-mutated erythroblasts may result not providing the episomal vectors. Furthermore, we acknowledge the only in mitochondrial iron overload, but also in the emer- support of Biomedical Research Core of Tohoku University School of gence of ferritin clumps as a consequence of cellular iron Medicine. overload. In support of our hypothesis, we have demon- Funding: this work was supported by JSPS KAKENHI Grant strated that Alas2-null murine definitive erythroblasts con- Number 16K19564 to SH. tain ferritin aggregates in the cytosol.5 Because our patient Correspondence: [email protected] with XLSA was responsive to vitamin B6, supplementing doi:10.3324/haematol.2017.179770 the culture media with vitamin B6 may partially restore the compromised ALAS2 activity enabling iron to be used cor- Information on authorship, contributions, and financial & other disclo- rectly. However, the differentiation of XiPS cells with vita- sures was provided by the authors and is available with the online version min B6-depleted IMDM medium did not induce ring sider- of this article at www.haematologica.org. oblasts (data not shown). It is anticipated that HCl pyridoxal supplementation of IMDM would be insufficient to References improve the enzymatic activity of the ALAS2 Val562Ala mutant. 1. Bottomley SS, Fleming MD. Sideroblastic anemia: diagnosis and management. Hematol Oncol Clin North Am. 2014;28(4):653-670. Initially, we applied a serum-free monolayer culture with 2. Ohba R, Furuyama K, Yoshida K, et al. Clinical and genetic charac- 11 the stepwise tuning of exogenous cytokine cocktails. teristics of congenital sideroblastic anemia: comparison with While this protocol has several advantages, including a sim- myelodysplastic syndrome with ring sideroblast (MDS-RS). Ann ple culture method without the need for stromal cells and Hematol. 2013;92(1):1-9. easy traceability of morphological changes,11 erythroblasts 3. Nakajima O, Takahashi S, Harigae H, et al. Heme deficiency in erythroid lineage causes differentiation arrest and cytoplasmic iron over- derived from XiPS cells did not exhibit ring sideroblasts. load. EMBO J. 1999;18(22):6282-6289. Although the reason remains unknown, we speculate that 4. Nakajima O, Okano S, Harada H, et al. Transgenic rescue of ery- stromal cells may promote erythroid differentiation in XiPS throid 5-aminolevulinate synthase-deficient mice results in the for- cells with the additional secreted factors from the stromal mation of ring sideroblasts and siderocytes. Genes Cells. cells or the signals delivered through adhesion molecules. 2006;11(6):685-700. 5. Harigae H, Nakajima O, Suwabe N, et al. Aberrant iron accumula- Besides the ALAS2 mutation, a series of factors critical for tion and oxidized status of erythroid-specific delta-aminolevulinate the formation of ring sideroblasts have been elucidated, synthase (ALAS2)-deficient definitive erythroblasts. Blood. including the mutation of genes involved in heme biosyn- 2003;101(3):1188-1193.

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