Dynamic Features of Chromosomal Instability during Culture of Induced Pluripotent Stem Cells

Abstract

Induced pluripotent stem cells (iPSCs) hold great potential for regenerative medicine. By reprogramming a patient′s own cells, immunological rejection can be avoided during transplantation. For expansion and gene editing, iPSCs are grown in artificial culture for extended times. Culture affords potential danger for the accumulation of genetic aberrations. To study these, two induced pluripotent stem (iPS) cell lines were cultured and periodically analyzed using advanced optical mapping to detect and classify chromosome numerical and segmental changes that included deletions, insertions, balanced translocations and inversions. In one of the lines, a population trisomic for chromosome 12 gained dominance over a small number of passages. This appearance and dominance of the culture by chromosome 12 trisomic cells was tracked through intermediate passages by the analysis of chromosome spreads. Mathematical modeling suggested that the proliferation rates of diploid versus trisomic cells could not account for the rapid dominance of the trisomic population. In addition, optical mapping revealed hundreds of structural variations distinct from those generally found within the human population. Many of these structural variants were detected in samples obtained early in the culturing process and were maintained in late passage samples, while others were acquired over the course of culturing.

Safety of GMP-compliant iPSC lines generated by Sendai virus transduction is dependent upon clone identity and sex of the donor

“…The frequency and probability of chromosomal aberrations in hiPSCs were dependent on both the iPSC line derived from the specific clone and the sex of the donor that the hiPSCs were generated; additionally it was higher in a woman. These conclusions were compared with existing literature data for both sexes, revealing a consistent trend [8,12,[16][17][18]22,26,[30][31][32][33]38,57]. The pluripotency of generated cells was primarily confirmed by the higher activity of alkaline phosphatase compared to fibroblasts.…”

“…The pluripotency of generated cells was primarily confirmed by the higher activity of alkaline phosphatase compared to fibroblasts. It is known that the high activity of alkaline phosphatase is considered a marker in ( HDFa-TK2) [12] of pluripotent stem cells [45]. Subsequently, immunocytochemistry revealed the presence of pluripotent markers OCT3/4, SOX2, and NANOG at the protein level, whereas analysis at the gene expression level allowed us to capture statistically significant differences in the OCT3/4 and NANOG expression between both sexes and hiPSC lines generated from the same donor.…”

CACNA1Ahaploinsufficiency leads to reduced synaptic function and increased intrinsic excitability

“…Copy number variation analysis by whole exome sequencing did not reveal any acquired variants in comparison to the published profile of this control line. 21…”

Optical Genome Mapping Reveals Genomic Alterations upon Gene Editing in hiPSCs: Implications for Neural Tissue Differentiation and Brain Organoid Research

“…Additionally, OMG enables the whole-genome characterization of previously undetectable aberrations [ 53 ]. Therefore, it has been recently used together with complementary quality control methods to assess the genome integrity of several hiPSC lines, identifying genomic alterations induced by reprogramming [ 56 ] and CRISPR/Cas9 genome editing [ 57 , 58 ], some of which were previously unrecognized [ 59 ], and to quantify and characterize chromosomal instability arising at different time points during iPSC culture [ 60 ]. On the other hand, the duo analysis approach, which relies on the normalization of the genomic data to a given data, can be used to reveal changes on a genomic level in hiPSCs before and after treatment/gene editing, accounting for the intrinsic genomic characteristics of the specific cell line.…”