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iPSC reprogramming-mediated aneuploidy correction in autosomal trisomy…
iPSC reprogramming-mediated aneuploidy correction in autosomal trisomy syndromes
Introduction
three types of live born trisomy
trisomy 21; Down syndrome, most common
trisomy 18; Edwards syndrome
trisomy 13; Patau syndrome, least common
chromosomal therapy
developed to correct an excess trisomic chromosome in cultured patient cells
to gain insights on trisomy rescure mechanisms; reprogrammed skin fibroblasts from patients with trisomy syndromes and analyzed iPSC colonies
results
Trisomy 13 rescue to disomy
obtained skin fibroblasts from three patients with trisomy 13
27 iPSC clones were generated
22 iPSC clones had disomy 13
6 iPSC clones remained trisomy 13
iPSC#22 had trisomy 13 mosaicism
performed single nucleotide polymorphism (SNP) genotyping analysis
20/22 iPSC clones had a combination of the 1st and 2nd chromosomes
iPSC#19 had the 1st and 3rd chromosomes
iPSC#15 had the 2nd and 3rd chromosomes
generated 22 iPSC clones from GM03330 skin fibroblasts
9/22 iPSC clones had disomy 13
4 clones had the 1st and 2nd chromosomes
5 clones had the 1st and 3rd chromosomes
Obtained skin fibroblasts from 2 patients with trisomy 21
1 iPSC clone had disomy 21
23 iPSC clones remained trisomy 21
Obtained skin fibroblasts from 2 patients with trisomy 18
iPSC clone with a combination of the 1st and 2nd chromosomes and two iPSC clones with a combination of the 1st and 3rd chromosomes all showed heterodisomy
iPSC clone with a combination of the 2nd and 3rd chromosomes showed segmental uniparental isodisomy
obtained skin fibroblasts from a patient with trisomy 9
20/21 metaphases were trisomy
1/21 were polyploid cells
performed single nucleotide polymorphism (SNP) genotyping analysis
5/15 clones had the 1st and 2nd chromosomes
6/15 clones had the 1st and 3rd chromosomes
4/15 clones had the 2nd and 3rd chromosomes
methods
obtained human fibroblast cell lines from Coriell institute for medical research
Human skin fibroblast cells were reprogrammed using integration-free episomal vectors carrying Oct-4, Sox2, Lin28, L-Myc, Klf4, mp53DD, and EBNA
Human skin fibroblasts (GM02948) and the iPSC clones generated were stained for key pluripotent markers using the PSC 4-marker immunocytochemistry kit
iPSCs were seeded in 35 mm dishes and incubated for 24 h in Essential 8 flex medium
After 24 h incubation, 0.04 μg/mL of KaryoMAXTM colcemidTM solution was added to arrest in metaphase for 2 hours
iPSCs were dissociated by 0.05% Trypsin-EDTA and treated with a hypotonic solution
Slides were prepared using an air-drying device for metaphase preparation
20 metaphases were examined in each cell line
Probes were prepared in FISH hybridization buffer FFPE and denatured for 6 min at 73°C
discussion
resuce efficiencies
Trisomy 21 syndrome cells showed the lowest trisomy rescue efficiencies, 4.2% for GM02967 and 0% for GM04616
Trisomy 18 syndrome cells showed efficiencies of 12.5% for GM03538 and 5.0% for GM00734
Trisomy 13 cells showed 81.5% for GM02948, 40.9% for GM03330 and 23.8% for GM00526
SNP array and sequencing analyses
revealed multiple combinations of chromosomes in several trisomy-rescued iPSC clones
suggests that the selection of chromosome pairs occurs randomly
established a total of 57 iPSC clones whose trisomy was rescued upon cell reprogramming
reported that anaphase lag mechanism may explain random loss of the trisomic chromosomes
demonstrated that trisomy rescue may randomly lose one copy of the trisomic chromosomes in the parental cells, resulting in three possible combinations of chromosome pairs
two possible mechanisms for trisomy rescue
pre-existing normal karyotype cells in the initial cell population are selected for iPSC; Ruled out because multiple combinations of chromosomes were found in trisomy-rescued iPSC clones
revertant cells occur spontaneously and are selected for because of a growth advantage during reprogramming