Safe harbor selection strategy and characterization of thal-iPS cell lines. (a) Following the establishment of patient-specific iPS cell lines, which in this study were generated from skin fibroblasts or bone marrow mesenchymal stem cells (BM MSCs) from β-thalassemia major patients, with an excisable single polycistronic vector co-expressing OCT4, KLF4, cMYC and SOX2 (illustrated in Supplementary Fig. 10a), the genetic rescue strategy is as follows: thal-iPS cells are transduced with a lentiviral vector expressing β-globin and an excisable Neo-eGFP selection cassette and subcloned into single cells, and single-vector-copy integrants are selected according to vector chromosomal position. The levels of β-globin expression afforded by the vector integrated at different genomic positions are analyzed in the erythroid progeny of each selected clone. Microarray analysis is used to examine perturbation of endogenous gene expression by the integrated provirus. The reprogramming vector can be efficiently excised before globin gene transfer or after—together with the Neo-eGFP selection cassette of the globin vector—through transient expression of Cre (Supplementary Fig. 10). (b) MSCs from β-thalassemia major patient 1. (c) Thal1.52 iPS cell line. (d) Expression of pluripotency markers in iPS cell lines thal1.52, thal2.1, thal5.10 and thal5.11. Thal1 MSCs: MSCs from β-thalassemia major patient 1; H1 hES: hES cell line H1. (e) NANOG expression in iPS cell lines. (f) Immunohistochemical analysis of a teratoma derived from line thal1.52. Upper panel: cytokeratin (CK) 20-positive intestine-like epithelium (endoderm); middle panel: vimentin-positive fibroblastic spindle cells (mesoderm); lower panel: S-100–positive peripheral nerve (ectoderm). (g) Bisulfite sequencing analysis of the OCT4 promoter in the indicated thal-iPS cell lines and the MSCs from which they were derived. Each horizontal row of circles represents an individual sequencing reaction with white circles representing unmethylated CpG dinucleotides and black circles representing methylated CpG dinucleotides. The numbers indicate the CpG position relative to the transcriptional start site (TSS). (h) Karyotype analysis of thal2.1 iPS cell line. Scale bars, 50 μm.

Single-vector copy, clonality and mapping of the integration site. (a,b) Upper panel: schematic representation of the TNS9.3/fNG lentiviral vector. An asterisk depicts a 4-bp insertion in the 5′ untranslated region (UTR) of the β-globin gene, which allows discrimination of the longer vector-encoded transcript from the endogenous β-globin transcript. TNS9.3/fNG also contains the human phosphoglycerate kinase (hPGK) promoter-driven neomycin phosphoryltransferase (Neo) and enhanced green fluorescent protein (eGFP) genes flanked by loxP sites. LTR: long terminal repeat; RRE: rev-responsive element; cPPT: central polypurine tract; HS: DNAse I hypersensitive site. Lower panels: Southern blot analysis to ascertain single integrations of the TNS9.3/fNG vector and clonality. Genomic DNA was digested with EcoRI (a) or XbaI (b). The probe used in a is eGFP (shown in the upper panel). The probe in b spans exons 1 and 2 of the β-globin gene (shown in the upper panel). The parental thal-iPS cell lines thal1.52, thal2.1, thal5.10 and thal5.11 and the clone number are depicted above the lanes. UT: untransduced. Arrowheads in a indicate bands corresponding to the reprogramming vectors pLM-GO, pLM-YS and pLM-CM (Supplementary Fig. 8d) present in the thal1.52 line and the clones derived from it. Arrowheads in b indicate endogenous bands (corresponding to the endogenous β-globin locus). Asterisks depict unique vector integration bands. (c–f) Examples of chromosome ideograms (upper panels) and graphics (lower panels) depicting 300 kb of human genome on both sides of the globin vector integration site in iPS clones thal1.52-10 (c), thal2.1-49 (d), thal1.52-17 (e) and the safe harbor clone thal5.10-2 (f). A vertical red line depicts the position of the vector insertion. Numbers depict positions in the corresponding human chromosome. All RefSeq genes present in the genomic region spanning 600 kb illustrated in the graphic are shown in blue. Genes implicated in cancer (Supplementary Table 1) are shown in red. Chromosome ideograms and graphics were generated with the UCSC Genome Graphs tool.

β-globin expression in the erythroid progeny of single-vector-copy thal-iPS cell clones. (a) Expression of erythroid cell markers CD71 and glycophorin A (GPA) in the erythroid progeny of thal-iPS cell line 1.52. (b) β-globin expression in the erythroid progeny of 13 single-vector-copy thal-iPS cell clones assessed by qRT-PCR. Expression levels are expressed per gene copy, relative to the average endogenous β-globin expressed in the in vitro differentiated erythroid progeny of peripheral blood CD34⁺ cells from four healthy individuals and normalized to endogenous α-globin expression. hES: erythroid progeny of hES cell line H1, wt iPS: erythroid progeny of iPS cell line FDCT, derived from fibroblasts of an 11-year-old healthy individual³⁰. Numbers below graphs depict thal-iPS clone numbers derived from lines thal1.52, thal2.1, thal5.10 and thal5.11. n: number of independent differentiations for each clone. UT: untransduced. Error bars denote s.e.m. (c) β-globin expression in the erythroid progeny of a subset of single-vector-copy thal-iPS cell clones and controls assessed by quantitative primer extension. β_E: endogenous β-globin (80 bp); α_E: endogenous α-globin (60 bp); β_V: vector-encoded β-globin (84 bp). PB CD34⁺: erythroid cell derivatives of in vitro differentiated peripheral blood (PB) CD34⁺ cells from a normal donor; H1 hES: erythroid cell derivatives of the H1 hES line; wt iPS: erythroid cell derivatives of iPS cell line FDCT, derived from fibroblasts of a healthy individual; thal1.52 UT, thal2.1 UT: erythroid cell derivatives of untransduced lines thal1.52 and thal2.1, respectively; thal1.52-17, thal2.1-67, thal1.52-10, thal1.52-16, thal1.52-38, thal2.1-49, thal2.1-48, thal2.1-55, thal5.11-32, thal5.10-2: erythroid cell derivatives of the respective single-vector-copy thal-iPS clones. (d) Chromatograms of HPLC analysis of α- and β-globin expression in the erythroid progeny of clones thal5.10-2 and thal5.11-28. Cord blood, H1 hES and untransduced (UT) thal1.52 cells were used as controls. For quantification of these data, see Supplementary Table 7.