The dark side of induced pluripotency

MARTIN F. PERA

Induced pluripotent stem cells (iPSCs) are generated through the reprogramming of differentiated adult cells and can be coaxed to develop into a wide range of cell types. They therefore have far-reaching potential for use in research and in regenerative medicine. But the ultimate value of these cells as disease models or as sources for transplantation therapy will depend on the fidelity of their reprogramming to the pluripotent state, and on their maintenance of a normal genetic and epigenetic (involving aspects other than DNA sequence) status. Recent surveys show that the reprogramming process and subsequent culture of iPSCs in vitro can induce genetic and epigenetic abnormalities in these cells. The studies raise concerns over the implications of such aberrations for future applications of iPSCs.

It has long been known that, during cultivation in vitro, human embryonic stem cells (ESCs) can become aneuploid; that is, they acquire an abnormal number of chromosomes. The new papers have applied various state-of-the-art genomic technologies to assess in detail the occurrence and frequency of genetic and epigenetic defects in both human iPSCs and ESCs.

Hussein et al. studied copy number variation (CNV) across the genome during iPSC generation, whereas Gore and colleagues looked for point mutations in iPSCs using genome-wide sequencing of protein-coding regions. Lister et al. examined DNA methylation — an epigenetic mark — across the genomes of ESCs and iPSCs at the single-base level. These studies, along with other investigations into changes in chromosome numbers and CNV in the two kinds of stem cell, lead to the conclusion that reprogramming and subsequent expansion of iPSCs in culture can lead to the accumulation of diverse abnormalities at the chromosomal, subchromosomal and single-base levels. Specifically, three common themes, regarding the genetic and epigenetic stability of ESCs and iPSCs, emerge.

First, by several measures, iPSCs display more genetic and epigenetic abnormalities than do ESCs or fibroblasts — the cells from which they originated. Chromosomal abnormalities appear early during the culturing of iPSCs5, a phenomenon not generally observed in ESCs. Also, the frequency of mutations in iPSCs is estimated to be ten times higher than in fibroblasts. And there are greater numbers of novel CNVs (CNVs not found in the cell of origin or in human genomes of comparable background) in iPSCs than in ESCs. Similarly, the epigenome of iPSCs features incomplete reprogramming (with cells retaining epigenetic marks of the cell of origin), aberrant methylation of CG dinucleotides, and abnormalities in non-CG methylation — an epigenetic feature seen only in pluripotent cells.

The research groups report clues to the potential function of the genetic lesions that arise in ESCs and iPSCs. For example, regions prone to amplification, deletion or point mutation seem to be enriched in genes involved in cell-cycle regulation and cancer. Although the changes observed do not strongly implicate any particular gene functionally as a target for change during the amplification of iPSCs or during their adaptation to culture conditions, the frequent association of the affected genes with cancer gives cause for concern.

With regard to evaluating the safety of ESCs and iPSCs, a key issue is the biological significance of the changes that these studies report. Clearly, aneuploid cell lines would not be used in therapy (although they might be useful for research into the basis of genetic disorders associated with anomalies in chromosome number or other genetic abnormalities). Cell lines bearing mutations of established functional consequence in oncogenes or tumour suppressors, or in genes associated with Mendelian disorders (those usually due to a single gene), could equally not be used therapeutically.

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