Generation And Functional Analysis Of Congenital Dyserythropoietic Anemia (Cda) Patient-Specific Induced Pluripotent Stem Cells

Abstract The congenital dyserythropoietic anemias (CDAs) are inherited red blood cell disorders representing ineffective erythropoiesis and dyserythropoietic changes in the bone marrow. We recently diagnosed a female patient with undiagnosed congenital anemia as type IV CDA caused by a heterozygous missense mutation of the erythroid-specific transcription factor, KLF1; c.973G>A, p. E325K. Although the mutation has been reported in a male patient characterized as hydrops fetalis, severe neonatal jaundice and transfusion-dependent anemia (Arnaud L et al., Am J Hum Genet, 2010), the proband showed relatively mild phenotype showing moderate dyserythropoietic anemia. In order to investigate the pathological significance of mutant KLF1 during erythroid cell development and differentiation, we generated induced pluripotent stem cells (iPSCs) from peripheral blood of the CDA patient (CDA-iPSCs), and utilized these cells to establish in vitro CDA model for better understanding of its molecular basis. CDA-iPSCs were generated from T lymphocytes in peripheral blood mononuclear cells. Hematopoietic precursors were induced from CDA-iPSCs by embryoid bodies formation. CD34(+) precursor cells were isolated and further cultured in liquid culture with cytokine cocktail (erythropoietin (EPO), interleukin (IL)-3, and stem cell factor (SCF)) for additional 1-3 weeks. Flow cytometric analysis showed that CDA-iPSC-derived cells contained significantly lower percentage of CD235a(+)/CD71(+) erythroid lineage cells than the cells derived from control iPSCs, and lack expression of the adhesion molecule CD44, which is known to be down regulated in peripheral blood erythroid cells of CDA patients (Arnaud L et al., Am J Hum Genet, 2010). In addition, colony-forming unit (CFU) assay indicated that CD34(+) fraction derived from CDA-iPSCs contained a lower number of erythroid colony-forming cells and the most of the cells in these colonies are morphologically abnormal, in comparison with control iPSCs. We next evaluated mRNA expression levels of fetal (HBG1 and HBG2), embryonic (HBE), and adult (HBB) globins, resulting that HBG1 and HBG2 were significantly increased in CDA-iPSCs-derived erythroid lineage cells, whereas HBE showed no significant change and HBB was decreased in CDA-iPSCs-derived erythroid lineage cells. However, BCL11A, one of the target genes of KLF1 and also known as a suppressor of HBG1 and HBG2, was not decreased in the presence KLF1 gene mutation, indicating that elevated HBG1 and HBG2 in CDA-iPSCs-derived erythroid cells was mediated by other mechanism like Leukemia/lymphoma Related Factor (LRF; Masuda T et al., Science, 2016). Here we suggest that our model provides insights on understanding the mechanisms of type IV CDA and the effect of KLF1 gene mutation on clinical phenotype and it would be a useful tool for drug screening and identification of novel biomarker for the rare congenital anemia. Figure 1 Induction of erythroid differentiation from human iPSCs Figure 2 Flow cytometric analysis of erythroid cells induced from CDA-iPSCs Figure 3 RT-PCR analysis of erythroid cells induced from CDA-iPSCs Disclosures Kohara: SBI Pharmaceuticals: Research Funding. Miyamoto:SBI Pharmaceuticals: Research Funding. Tani:Oncolys BioPharma: Equity Ownership; SymBio Pharmaceuticals: Consultancy, Equity Ownership; SBI Pharmaceuticals: Research Funding; Shinnihonseiyaku: Research Funding.