Extended Data Fig. 10: Analysis of patient and rescue iPS cell line-derived PSM and allele-specific gene correction of SCDP1 patient iPS cell lines.
From: Recapitulating the human segmentation clock with pluripotent stem cells
a, Representative DLL1 expression in iPS cells (grey) and PSMs (red) derived from a patient with SCD with a compound mutation in MESP2 (SCDP1-A and SCDP1-F), a patient with SCD with a mutation in DLL3 (SCDP2-A and SCDP2-E) and corresponding isogenic rescue cell lines (SCDP1-resA1, SCDP1-resF3, SCDP2-resA12 and SCDP2-resE17). n = 3; data for SCDP1-A and SCDP2-E are also used for Fig. 4b. b, Three-dimensional synchronization assay of SCDP1 patient PSM. Representative kymograph of three independent experiments is shown. c, Representative measurement of HES7 reporter activity in PSM derived from SCDP1 patient cell line. After the spike noise was removed, the signal of the entire area was averaged. The signal was further detrended and normalized to the average (±100-min window). d, Top, representative genotype of patients with SCD and iPS cells (SCDP1) with compound heterozygous mutations in MESP2. Bottom, sequence of each haplotype from patient genomic DNA. Red triangle indicates a deletion. Black triangle indicates a single nucleotide variation. e, Schematic of the gene-targeting procedure for allele-specific correction of MESP2 mutations using MhAX. Details of the targeting and genotyping procedures are provided in g. f, Genotype of heterozygously corrected iPS cell subclones. 201B7 is included as a reference. Red triangle indicates a deletion. Black triangle indicates a single nucleotide variation. DNA sequencing was performed twice for each clone; n = 2. g, Detailed schematic of gene-correction strategy of SCDP1 patient iPS cell clones. Depicted are two mutant or corrected MESP2 alleles with coding and non-coding exons (grey and white), overlapping donor vector homology arms (HA-L and HA-R), engineered 51-bp microhomology (μ51, blue), inverted protospacers for cassette excision (ps1, green), genotyping primers (red arrows) and Southern blotting probes (black bars). Sequences of mutation-specific sgRNAs are shown below each mutant allele. The gene-targeted intermediate shows details of the CAG::mCherry-IRES-puro cassette used for enrichment. h, Southern blot analysis of targeted iPS cell clones. Samples marked with an asterisk were selected for cassette excision. i, Southern blot analysis of gene-corrected iPS cell clones following selection marker removal. Samples marked with an asterisk were selected for phenotyping (067-1-3, SCDP1-resA1; 067-2-5, SCDP1-resF2; 067-3-4 and SCDP1-resF3). Southern blots shown in h and i were performed once for two patient and rescue clones each. For gel source data of h and i see Supplementary Fig. 1. j, k, Resulting karyograms from SNP array analysis of SCDP1 patient iPS cell clone A (SCDP1-A) and corresponding rescued iPS cell line (SCDP1-resA1). l–n, Karyograms from SNP array analysis of iPS cell clone F (SCDP1-F) from a patient with SDV and corresponding rescued iPS cell lines (SCDP1-resF2/F3). No de novo CNVs were detected following gene editing and subcloning. These figures were created with Illumina Genome Viewer (v.1.9.0) on Illumina GenomeStudio v.2011.1 with Human:Build 37 genome.
