title: Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients.
abstract: Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the "safe harbor" AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells.
authors.affiliation: Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
authors.forename: Loïc
authors.initials: L
authors.lastname: Garçon
authors.forename: Jingping
authors.initials: J
authors.lastname: Ge
authors.forename: Shwetha H
authors.initials: SH
authors.lastname: Manjunath
authors.forename: Jason A
authors.initials: JA
authors.lastname: Mills
authors.forename: Marisa
authors.initials: M
authors.lastname: Apicella
authors.forename: Shefali
authors.initials: S
authors.lastname: Parikh
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Sullivan
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Podsakoff
authors.forename: Paul
authors.initials: P
authors.lastname: Gadue
authors.forename: Deborah L
authors.initials: DL
authors.lastname: French
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Mason
authors.forename: Monica
authors.initials: M
authors.lastname: Bessler
authors.forename: Mitchell J
authors.initials: MJ
authors.lastname: Weiss
mesh_terms: D029503:Anemia, Diamond-Blackfan / Q000097:blood*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D019070:Cell Lineage; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D016086:Lentivirus / Q000235:genetics; D009154:Mutation; D012337:RNA, Ribosomal, 18S / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics; D054683:Ribosome Subunits, Large, Eukaryotic / Q000378:metabolism / Q000473:pathology; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism / Q000473:pathology; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D012337:RNA, Ribosomal, 18S; D012269:Ribosomal Proteins; C549166:ribosomal protein L5, human; C040343:ribosomal protein S19