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

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 (Knockout^TM 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.