Temporal Analysis of Genome Alterations Induced by Single-Cell Passaging in Human Embryonic Stem Cells

Abstract

Simplified culture conditions are essential for large-scale drug screening and medical applications of human pluripotent stem cells (hPSCs). However, hPSCs [ie, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (iPSCs) are prone to genomic instability, a phenomenon that is highly influenced by the culture conditions. Enzymatic dissociation, a cornerstone of large-scale hPSC culture systems, has been reported to be deleterious, but the extent and the timeline of the genomic alterations induced by this passaging technique are still unclear. We prospectively monitored three hESC lines that were initially derived and cultured on human feeders and passaged mechanically before switching to enzymatic single-cell passaging. We show that karyotype abnormalities and copy number variations are not restricted to long-term culture, but can occur very rapidly, within five passages after switching hESCs to enzymatic dissociation. Subchromosomal abnormalities preceded or accompanied karyotype abnormalities and were associated with increased occurrence of DNA double-strand breaks. Our results indicate that enzymatic single-cell passaging can be highly deleterious to the hPSC genome, even when used only for a limited period of time. Moreover, hPSC culture techniques should be reappraised by complementing the routine karyotype analysis with more sensitive techniques, such as microarrays, to detect subchromosomal abnormalities.

Methods of Reprogramming to Induced Pluripotent Stem Cell Associated with Chromosomal Integrity and Delineation of a Chromosome 5q Candidate Region for Growth Advantage

“…Two of these clones (L-K2A and S-K2A), generated by either reprogramming method, showed a chromosome 5 trisomy. Interestingly, a third line (L-K2B) showed a duplication of chromosome 5q13q33 in 9/20 cells (46,XY,dup(5)(q13q33) [9]/46,XY [11]). The duplication was detected already at P15 and after induction with lentivirus.…”

“…Furthermore, insertion of some vectors into the genome of reprogrammed cells is a concern as reactivation of the transgenes may result in oncogenesis [7]. However, the frequency of abnormalities in iPSC and embryonic stem cell (ESC) is similar, suggesting that reprogramming itself is not a significant source of rearrangement [4,5,8,9]. Furthermore, gain of chromosomes 12 and 20 is a common abnormality in both types of stem cells [4,5].…”

Induced Pluripotent Stem Cell Therapies for Degenerative Disease of the Outer Retina: Disease Modeling and Cell Replacement

“…34 Analyses on different hESC lines revealed that single-cell enzymatic passage can be deleterious even at early passages due to the increase in DNA double-strand breaks. 35 In fact, labor-intensive mechanical passage on feeders appears most ''protective'' for the cells, even up to 100 passages. [36][37][38] When directly compared, bulk enzymatic passaging methods of medium-size clumps of cells did not cause genomic aberration in the first 15 passages.…”

Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts

“…Several media supplements and cloning approaches have been developed to overcome this difficulty but are still fraught with either expensive equipment (low oxygen incubators), difficult technological steps (survival of FACS sorted single iPSCs), or labor intensive protocols (sub-cloning) (Forsyth et al ., 2006; Miyaoka et al ., 2014). Moreover, single cell passaging has been linked to increased genomic abnormalities in iPSCs (Bai et al ., 2015). Fluorescent or antibiotic resistance genetic markers have been used to overcome issues with clonality and the overall low efficiency of gene-editing in these cells, but require homologous recombination of a large cassette via a targeting plasmid designed with long homology arms (400–800 bp) (Hendel et al , 2014).…”