HEMATOPOIESIS AND STEM CELLS Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients

Diamond Blackfan anemia (DBA; OMIM105650) is a rare congenital anemia with erythroid (Ery) hypoplasia, developmental abnormalities, growth retardation, and an increased risk of malignancy. About half of DBA cases are caused by heterozygous mutations in genes encoding ribosomal proteins, leading to haploinsufficiency or, less frequently, dominant-negative effects with consequent defects in ribosome formation and/or function. RPS19 and RPL5 mutations are the most frequently mutated genes, representing ;25% and 7% of all cases. RPS19 is required for ribosomal RNA (rRNA) processing and assembly of the small 40S ribosomal subunit. RPL5 binds 5S rRNA thereby facilitating its transport to the nucleolus for assembly into the 60S ribosomal subunit. How ribosomal defects produce the unique constellation of DBA abnormalities is not understood. RPS19-deficient animal models, including genetically manipulated zebrafish and mice, provide insights into the pathophysiology of DBA, although the extent to which they recapitulate the hematopoietic defect in humans is not clear. The Ery defects of DBA have been characterized further through RPS19 knockdown in normal hematopoietic cells and examination of primary hematopoietic progenitors from affected patients. The pathophysiology of RPL5 mutations is largely unstudied and no relevant animal models are available, although this represents a relatively common DBA gene associated with a particularly high incidence of craniofacial, heart, and thumb malformations. Studies of affected tissues from DBA patients complement animal model studies. However, obtaining patient-derived DBA cells is limited by the rarity of the disease (especially non-RPS19 mutant forms), and the invasive nature of tissue sampling. Moreover, Ery progenitors are reduced in at least some forms of DBA due to enhanced apoptosis, which precludes obtaining the relevant affected cell type directly from patients. Consequently, adequate human cellular models for DBA are insufficient. Findings that human somatic tissues can be reprogrammed into induced pluripotent stem cells (iPSCs) via ectopic expression of several transcription factors have created new opportunities for disease modeling. iPSCs, which resemble embryonic stem cells (ESCs), can be differentiated to form blood lineages in vitro, and therefore offer potentially relevant tissue sources for studies of inherited blood disorders, including DBA. Moreover, patient-derived iPSCs are amenable to genetic manipulation and gene correction, providing a promising strategy for cellular therapy. We generated iPSCs from 2 DBA patients, 1 carrying a RPS19 mutation and the