Fig 1.
Trisomy 13 of GM02948 skin fibroblasts derived from a Patau syndrome patient was rescued to disomy by cell reprogramming.
(A) Scheme of an experimental procedure. Skin fibroblast lines at passage 2 were reprogrammed and the resultant iPSC colonies were picked up (passage 1) and examined for trisomy rescue at passage 2 by interphase fluorescence in situ hybridization (FISH) and karyotype analysis. (B) Interphase FISH analysis using chromosome 13 centromere enumeration probe (CEP13) Orange-Red. DAPI staining shows nucleus (blue). Both the skin fibroblast line and the resultant iPSC clone #23 show trisomy 13 (three red signals), while the iPSC clone #15 shows disomy 13 (two red signals). The graph below shows the interphase FISH results of trisomy and disomy in the skin fibroblasts and the isogenic iPSCs, with the karyotype results on the right. (C) Both skin fibroblasts and the iPSC clone #23 show a male karyotype with trisomy 13 in 20 out of 20 metaphases, while the iPSC clone #15 was trisomy-rescued to show a normal male karyotype in 20 out of 20 metaphases. (D) Array CGH profile for chromosome 13 showing an increased copy number of 0.5 in the fibroblasts, indicating a gain of chromosome 13, while a copy number of zero was observed in iPSC clone #15, indicating no imbalance for chromosome 13 content.
Table 1.
Numerical alteration for chromosomes in the cells with autosomal trisomies after cell reprogramming*.
Fig 2.
Single nucleotide polymorphism (SNP) analysis detected three different combinations of chromosome 13 pairs in the isogenic disomic iPSC clones.
(A) SNP profiles of GM02948 skin fibroblasts and their isogenic iPSC clones; #23, #7, #19, and #15. The fibroblasts and the non-rescued clone #23 both show full trisomy 13, while the trisomy-rescued clones #7, #19, and #15 show heterodisomy of chromosome 13. (B) Classification of the trisomy-rescued iPSC clones into three groups by the SNP genotyping data. Three SNPs (exm2267608-rs1992744; Chr13:75993887-rs9530435, and exm2267625-rs9514690) were selected and the nucleotides were determined by Sanger sequencing. The fibroblasts and iPSC clone #23 both show SNPs of all three different chromosomes, while iPSC clones #7, #19, and #15 show SNPs of just two different chromosomes: The 1st and 2nd, 1st and 3rd, and 2nd and 3rd, respectively.
Table 2.
Short tandem repeat (STR) analysis of skin fibroblasts GM02948 and their isogenic iPSC clones.
Fig 3.
SNP and Sanger sequence indicated two combinations of chromosomes 13 pairs in GM03330 disomy iPSC clones.
(A) Karyotype analysis of skin fibroblasts and the iPSC clone #23 both showed male trisomy 13. The iPSC clones #4 and #1 both showed trisomy-rescued disomy 13. (B) SNP analysis of skin fibroblasts P3 and the iPSC clone #23 both showed trisomy. In contrast, the iPSC clone #4 showed heterodisomy, and the iPSC clone #1 showed two segments of isodisomy (indicated in light green). (C) Sanger sequencing assessment of two different SNPs located on chromosome 13 (exm2260179- rs9510171, and Chr13:75993887- rs9530435) indicated a combination of the 1st and 2nd chromosomes in four iPSC clones; and a combination of the 1st and 3rd chromosomes in five iPSC clones. The UPiD is indicated by (**) and heterodisomy (*).
Table 3.
Chromosome-pair selection in trisomy-rescued iPSC clones.
Fig 4.
Trisomy rescue of chromosomes 21, 18, and 9 was detected by FISH and confirmed by karyotyping.
(A) The left graph shows the interphase FISH results of trisomy and disomy in skin fibroblasts from a Down syndrome (DS) patient (GM02767) and 24 isogenic iPSCs, with karyotype results on the right side of the graph. Trisomy 21 is shown in blue and disomy 21 in orange. The right side shows the karyotype results of the GM02767 skin fibroblasts, the non-rescued iPSC clone #2, and the trisomy-rescued iPSC clone #1. (B) The left graph shows the FISH results of GM03638 skin fibroblasts from a patient with Edwards syndrome and 32 iPSC clones (trisomy shown in blue and disomy in orange). The right side shows the karyotype results of the GM03538 fibroblasts, the non-rescued iPSC clone #16, and the trisomy-rescued iPSC clone #1. (C) The left graph shows the FISH results of GM09286 skin fibroblasts from a patient with trisomy 9 syndrome and 33 iPSC clones (trisomy shown in blue, disomy in orange, polyploidy: Four spots in gray and six spots in yellow). The right side shows the karyotype results of the GM09286 fibroblasts, the non-rescued iPSC clone #17, and the trisomy-rescued iPSC clone #12. Polyploid cells in the GM09286 fibroblasts were not included in the karyotype results.
Fig 5.
Proposed model of iPSC reprogramming-mediated trisomy rescue.
(A) Random loss of trisomic chromosome occur after induction of pluripotency by expression of OSKML (Oct4, Sox2, Klf4, L-myc and Lin28) genes. Disomic cells are then selected against trisomic cells. T = trisomy; D = disomy. (B) Random loss of trisomic chromosome is mainly due to anaphase lagging.