September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Assessment of pluripotent stem cell clonality and virtual karyotype, of cells from patients with eye disease, using low-density SNP arrays.
Author Affiliations & Notes
  • Alex W Hewitt
    Centre for Eye Research Australia, Melbourne, Victoria, Australia
    University of Tasmania, Hobart, Tasmania, Australia
  • Duncan E Crombie
    Centre for Eye Research Australia, Melbourne, Victoria, Australia
  • Kathryn Davidson
    Centre for Eye Research Australia, Melbourne, Victoria, Australia
  • Helena Liang
    Centre for Eye Research Australia, Melbourne, Victoria, Australia
  • Ray Wong
    Centre for Eye Research Australia, Melbourne, Victoria, Australia
  • Sandy Shen-Chi Hung
    Centre for Eye Research Australia, Melbourne, Victoria, Australia
  • Sook Chung
    Save Sight Institute, Sydney, New South Wales, Australia
  • Alice Pébay
    Centre for Eye Research Australia, Melbourne, Victoria, Australia
  • Footnotes
    Commercial Relationships   Alex Hewitt, None; Duncan Crombie, None; Kathryn Davidson, None; Helena Liang, None; Ray Wong, None; Sandy Hung, None; Sook Chung, None; Alice Pébay, None
  • Footnotes
    Support  Peter and Joan Clemenger Trust; NHMRC 1059369
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 6063. doi:
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      Alex W Hewitt, Duncan E Crombie, Kathryn Davidson, Helena Liang, Ray Wong, Sandy Shen-Chi Hung, Sook Chung, Alice Pébay; Assessment of pluripotent stem cell clonality and virtual karyotype, of cells from patients with eye disease, using low-density SNP arrays.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6063.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Prior to using human pluripotent stem cells for in vitro or in vivo applications, they must undergo rigorous characterisation to ensure they are representative of pluripotent stem cells and verify that have not incorporated new genomic aberrations. Here, we investigated the utility of a low-density genome-wide SNP array for the karyotypic assessment of induced pluripotent stem cells (iPSCs) using the Illumina Infinium HumanCore BeadChip.

Methods : Experimental work performed in this study was approved by the IRB of the RVEEH and we conformed with the Declarations of Helsinki. Primary human dermal fibroblasts were isolated from skin punch biopsy specimens of a patient (MT1) with idiopathic juxtafoveal macular telangiectasia using standard techniques. iPSC lines MT1, the hESC line H9 and the abnormal hESC line BG01V (49, +12, +17, XXY) were maintained undifferentiated. Following standard G-banding cytogenetic karyotyping, normal and abnormal iPSC clones generated from MT1 were pooled at specific ratios. Genomic DNA extracted from each pool was hybridised to Infinium HumanCore Arrays and probes passing the initial QC were used for copy number variant (CNV) assessment. CNV analysis was performed using PennCNV and QuantiSNP. Assessment of clonality was performed using CHAT.

Results : G-banding revealed chromosomal aberration in 39 of 50 metaphases of MT1-CL5 (mos 46,XX,add(5)(p15.3)[39]/46,XX[11]) and a normal cytogenetic karyotype for MT1-CL8. Array-based genotyping showed that MT1-CL5 had the highest number of discordant genotypes. Virtual karyotyping DNA extracted from pools with different ratios of these normal (MT1-CL8) and aberrant clones (MT1-CL5) revealed that we were able to detect abnormalities when at least 39% of the cell population was abnormal.

Conclusions : Our data demonstrates that an array-based karyotyping offers an economical and robust sampling method, both in the number of cells studied and genomic resolution, compared with standard cytogenetic karyotyping of hPSCs. This approach could provide a reliable, rapid and cost effective assessment of pluripotent stem cell clonality and virtual karyotype for the large scale generation and maintenance of hPSCs for a variety of eye diseases.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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