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Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
STEM_749_sm_supplMethods.pdf26KSupplementary Data
STEM_749_sm_supplDiscussion.pdf12KSupplementary Data
STEM_749_sm_supplTable1.tif71KSupplementary Table 1.
STEM_749_sm_supplTable2.tif70KSupplementary Table 2.
STEM_749_sm_supplFig1.tif161KFigure S1. Detection of SMN1 gene defect in GM09677 fibroblasts. (A) The genomic DNA was prepared from the cell line indicated. Exon 7 was amplified by PCR and the product digested with Dra1. Dra1 digestion of the PCR product should generate two fragments (arrows). The upper fragment represents the SMN1 gene resistant to Dra1 digestion and the lower fragment represents the SMN2 gene which was digested into two fragments with the smaller fragment running off the gel. (B) Exon 8 was amplified by PCR and the product digested with Dde1. The upper fragment represents the SMN1 gene resistant to Dde1 digestion and the lower two fragments represent the digested product of the SMN2 gene (arrows). The analysis suggested that, similar to other type 1 SMA patients, GM09677 had a homozygous deletion in the SMN1 gene that spanned at least most of the exon 7 and exon 8. IMR-90: normal human fibroblasts; GM09677: SMA fibroblasts; SMA-23 to SMA-27: iPS cell lines derived from GM09677 fibroblasts.
STEM_749_sm_supplFig2D-E.tif190KFigure S2. Characterization of SMA iPS cell lines. (A) The morphology of a typical SMA iPS cell colony. (B) Immunofluorescence detection of Tra1-81 expression in SMA iPS cells. (C) RT-PCR analysis of ES cell-specific gene expression in SMA iPS cell lines. Primers used for oct3/4, sox2, klf4 and c-myc detect only the transcripts of the endogenous genes, not the transgenes. RNAs isolated from GM09677 fibroblasts and H9 cells served as the negative and positive controls respectively for ES cell-specific gene expression. Primers used for RT-PCR are shown in Table S1. (D) SMA iPS cells maintain a normal karyotype. SMA-23 cells were passaged ten times prior to karyotype analysis. Metaphase chromosomes from a total of 26 cells were counted with a distribution of 2 cells containing 45 chromosomes, 22 cells containing 46 chromosomes and 2 cells containing 47 chromosomes. (E) Quantitative RT-PCR analysis of retroviral transgene expression in SMA iPS cells. Total RNA was prepared from the cell indicated; F: GM09677 fibroblasts; ES: HES2 cells, a well-established human ES cell line; numbers denote SMA iPS cell clone numbers as described in the main text. Two primer pairs for each gene were used: one to amplify the total transcript (filled bars) and the other to amplify the endogenous transcript (open bars). The value of each gene in individual cell lines was normalized against an internal control (GAPDH) and plotted relative to the value in HES2. The data is presented as mean ± SD.
STEM_749_sm_supplFig2A-2C.tif465KFigure S2. Characterization of SMA iPS cell lines. (A) The morphology of a typical SMA iPS cell colony. (B) Immunofluorescence detection of Tra1-81 expression in SMA iPS cells. (C) RT-PCR analysis of ES cell-specific gene expression in SMA iPS cell lines. Primers used for oct3/4, sox2, klf4 and c-myc detect only the transcripts of the endogenous genes, not the transgenes. RNAs isolated from GM09677 fibroblasts and H9 cells served as the negative and positive controls respectively for ES cell-specific gene expression. Primers used for RT-PCR are shown in Table S1. (D) SMA iPS cells maintain a normal karyotype. SMA-23 cells were passaged ten times prior to karyotype analysis. Metaphase chromosomes from a total of 26 cells were counted with a distribution of 2 cells containing 45 chromosomes, 22 cells containing 46 chromosomes and 2 cells containing 47 chromosomes. (E) Quantitative RT-PCR analysis of retroviral transgene expression in SMA iPS cells. Total RNA was prepared from the cell indicated; F: GM09677 fibroblasts; ES: HES2 cells, a well-established human ES cell line; numbers denote SMA iPS cell clone numbers as described in the main text. Two primer pairs for each gene were used: one to amplify the total transcript (filled bars) and the other to amplify the endogenous transcript (open bars). The value of each gene in individual cell lines was normalized against an internal control (GAPDH) and plotted relative to the value in HES2. The data is presented as mean ± SD.
STEM_749_sm_supplFig3.tif208KFigure S3. DNA fingerprint analysis for the origin of SMA iPS cell lines. The results from two SMA iPS cell lines, SMA-23 and SMA-26, are shown. Jurkat is a human T cell lymphoma line, 293T is a human kidney epithelial line and IMR-90 is a normal human fibroblast line. The name of each primer pair for the amplification of short tandem repeats (STRs) in the genomic DNA is given in each gel. PCR products were separated in 4% NuSieve gels. The whole set of primer sequences for STR analysis is listed in Table S2. The STR study confirmed that the two SMA iPS cell lines were derived from GM09677 fibroblasts, and other SMA iPS cell lines showed identical results (not shown). These studies confirmed that the established SMA iPS cell lines were indeed derived from the parental GM09677 fibroblasts, and not from the contamination of other cell lines grown in the laboratory.

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