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Induced Pluripotent Stem Cell Variability Tallied Across Genomes

NEW YORK (GenomeWeb) – Members of an international team have taken a closer look at the variation that exists in induced pluripotent stem cells (iPSCs) — and the potential causes and consequences of such dissimilarities.

"[C]ommon genetic variants produce readily detectable effects in iPSCs and provide the most comprehensive map of regulatory variation in human iPSCs to date," Sanger Institute investigator Daniel Gaffney and European Molecular Biology Laboratory researcher Oliver Stegle, corresponding authors on today's Nature study, and their colleagues wrote.

"We also demonstrate that differences between donor individuals have pervasive effects at all phenotypic levels in iPSCs, from the epigenome, transcriptome, and proteome to cell differentiation and morphology," they added.

As part of the Human Induced Pluripotent Stem Cells Initiative, the researchers systematically profiled genotyping patterns and related phenotypes in 711 iPSC lines that had been generated from 301 apparently healthy individuals.

Their results indicated that anywhere from 5 percent to nearly half of the phenotypic variability present in iPSC lines can be traced back to the underlying genetic differences that exist from one individual to the next. While they did see some rare, recurrent copy number alterations in the iPSC lines, genetic anomalies were not as widespread as indicated by some prior iPSC studies.

"Compared to previous work, we observed substantially lower levels of genetic aberrations," the authors wrote. "One possible explanation is that access to donor-matched reference samples helped us more accurately identify germline [copy number alterations] that would otherwise have inflated our estimates."

Though many researchers are optimistic about the possibilities of applying iPSC-based approaches to understanding and perhaps treating some diseases, the authors noted, "variable genetic and phenotypic characterization of many existing iPSC lines limits their potential use for research and therapy."

Using fibroblasts obtained from skin punch samples for 301 donor individuals, the team established 711 iPSC lines — a set that included at least two lines apiece for 82 percent of the participants and additional cell lines for roughly half of the individuals.

These iPSC lines and fibroblast samples were then subjected to array-based genotyping and expression profiling, along with other assays aimed at untangling cells' differentiation potential and pluripotency.

The investigators also did more extensive phenotyping and molecular analyses — including proteomic and array-based DNA methylation profiling — on a subset of the samples. And to get a glimpse at the features found in differentiated cells, they turned to immunohistochemistry and imaging, comparing hundreds of iPSC lines to almost 400 lines nudged to differentiate into neuroectoderm, meosoderm, or endoderm cells.

The results suggested more than 80 percent of the iPSC lines were indeed pluripotent, for example, while anywhere from 18 to 62 percent of cells per line expressed three pluripotency marker genes. Likewise, differentiation typically produced some 70 to 84 percent of cells, on average, that expressed appropriate germ layer markers.

On the genotype and phenotype side, the team's results revealed copy number changes in more than 40 percent of the lines, regardless of donor age, gender, or cell passage number. Those alterations tended to turn up at almost three-dozen recurrent sites in the genome.

A smaller subset of the lines — 4 percent — contained detectable trisomies, the researchers reported, although lines established from the same donor individual tended to have reproducible molecular and phenotypic features.

The team's analyses offered a more comprehensive look at expression quantitative trait loci (eQTL), too. With RNA sequencing-based expression profiles for lines generated from 166 unrelated participants, the group profiled iPSC-specific eQTLs and secondary eQTLs alongside eQTLs from somatic tissue to gauge their prevalence, effects, and potential contributions to disease (based on overlap with disease-associated variants).

"We have identified eQTLs that function primarily in pluripotent cells, a subset of which tag loci associated with disease," the authors wrote. "These loci may drive disease susceptibility through molecular changes early in development or, more generally, in cells with 'stem-like' characteristics, which are not well captured by studies of differentiated primary tissues from adult individuals."