The

n e w e ng l a n d j o u r na l

of

m e dic i n e

clinical implications of basic research

Induced Pluripotent Stem Cells A Cautionary Note

Christine Mummery, Ph.D. In 2006, when Shinya Yamanaka found that introducing just four genes into normal mouse skin cells made those cells pluripotent and thus able to generate all cell types of the body the reaction was almost one of disbelief. But less than a year later, both he and James Thomson did the same with human cells. Human pluripotent stem cells could not only be obtained without the use of embryos, but they could also be derived from any individual of choice. These advances had decidedly positive implications for cell therapy and regenerative medicine at least, perhaps, until now. Three recent studies have shown that genetic abnormalities (affecting DNA) and epigenetic abnormalities (not directly affecting DNA) arise during reprogramming or in subsequent cell culture.1-3 In these studies the investigators used different state-of-the-art molecular techniques (ones that measure variation in the copy number of genes and associated DNA, point mutations in protein-coding regions, and DNA methylation of single bases) to examine different human induced pluripotent stem (hiPS) cell lines and to determine when lesions arose, which genes might be affected, and whether the defects resembled those described in other cultured cells or stem cells derived from human embryos, also known as human embryonic stem (hES) cells. The results of these studies are consistent with other findings and show that hiPS cells accumulate chromosomal, subchromosomal, and single-base changes over time, some of which are the same as those described in hES cells and human embryonal carcinoma cells.4 These changes include duplications in parts of chromosomes 12 and 20 and the failure of some regions of the genome to fully reprogram from the differentiated to the undifferentiated state, leaving characteristic marks of the original fibroblasts in the derivative hiPS cell lines. Although the consequences of these particular changes remain un2160

known, the changes are fairly easily detected and therefore might not represent real risk, since cell lines that exhibit such changes could be excluded from clinical use. Of greater concern are the more subtle changes and point mutations that may affect genes essential for cell growth, tumor suppression, or other critical functions. In fact, the studies suggested that regions of the genome prone to amplification, deletions, or point mutations were also regions encoding many genes associated with cell-cycle control and cancer. Most striking was the finding that although some of the lesions were inherited from a small minority of the original fibroblasts, many more lesions accumulated during reprogramming (Fig. 1). Oddly, many of these disappeared during prolonged culture, a finding that suggests that they might be deleterious for the subpopulation in which they occur. So why the cautionary note? As long as we do not know whether these barely detectable changes have any functional significance, it would probably be unwise to use the cells clinically. All truly pluripotent stem cells, such as both hES and hiPS cells, have an intrinsic ability to form teratomas after transplantation, so any residual undifferentiated cells compromise safety. At issue here, though, are any other forms of abnormal behavior that arise from the lesions introduced as a result of reprogramming. Although hiPS cells can be made from any individual patient and can thus, in principle, be used autologously, this personalized form of therapy would probably be extraordinarily expensive in practice because of the high costs associated with safety checks and cell production. Rather, it is generally expected that cell banks, much like those used for umbilical-cord blood, will be established from which clinicians could tap cells with an HLA match for their particular patient. If assays could be established (on the basis of techniques described in the recent reports), hiPS cells could be screened before cell-

n engl j med 364;22

nejm.org

june 2, 2011

The New England Journal of Medicine Downloaded from nejm.org on September 28, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.

clinical implications of basic research

Figure 1. Genetic Effects of Reprogramming Cells for Pluripotency. Genetic lesions arise during reprogramming of fibroblasts to pluripotency (thus generating human induced pluripotent stem [hiPS] cells) and during prolonged culture of both hiPS cells and human embryonic stem (hES) cells. Recent studies show that in addition to gross chromosomal changes that occur during prolonged culture of hiPS and hES cells (e.g., duplication of parts of chromosomes 12 and 20), gene copy-number variations and point mutations can be induced during the reprogramming of somatic cells into hiPS cells, resulting in many more DNA lesions (by up to a factor of 10) in hiPS cells than in the somatic cells from which they are derived.1-5 During prolonged culture, the frequency with which these new mutations are detected decreases.

bank deposition so that cell lines could be selected with some degree of assurance that they do not carry mutations. A more immediate application of hiPS cells is their use in modeling human disease. For example, there are several reports of patient-specific hiPS cell lines with inherited mutations in ionchannel genes that cause sudden cardiac death as a result of an abnormal heartbeat.5 Cardiomyocytes obtained by inducing differentiation of these hiPS cell lines show disease features similar to those of the patients from whom they

were derived, including enhanced sensitivity to specific drugs. But are the phenotypes in cardiomyocytes derived from hiPS cells really attributable to the mutation in the patient and not to some other mutation introduced during reprogramming? The demonstration that the disease phenotype can be genetically rescued may soon be a required control. Removal of the mutant allele, overexpression of the wild-type gene, or replacement of the mutation by means of gene targeting would be the most effective strategy to mediate rescue and prove cause and effect.
2161

n engl j med 364;22

nejm.org

june 2, 2011

The New England Journal of Medicine Downloaded from nejm.org on September 28, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.

clinical implications of basic research

The discovery of direct reprogramming remains a major breakthrough, but we may now need to redouble efforts to get hiPS cells one step closer to hES cells (which are still the bestcharacterized pluripotent cells) in order to exploit hiPS cells to the fullest.
Disclosure forms provided by the author are available with the full text of this article at NEJM.org. From the Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, the Netherlands.

1. Hussein SM, Batada NN, Vuoristo S, et al. Copy number

variation and selection during reprogramming to pluripotency. Nature 2011;471:58-62. 2. Gore A, Li Z, Fung HL, et al. Somatic coding mutations in human induced pluripotent stem cells. Nature 2011;471:63-7. 3. Lister R, Pelizzola M, Kida YS, et al. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 2011;471:68-73. 4. Pera MF. Stem cells: the dark side of induced pluripotency. Nature 2011;471:46-7. 5. Dambrot C, Passier R, Atsma D, Mummery CL. Cardiomyocyte differentiation of pluripotent stem cells and their use as cardiac disease models. Biochem J 2011;434:25-35.
Copyright 2011 Massachusetts Medical Society.

2162

n engl j med 364;22

nejm.org

june 2, 2011

The New England Journal of Medicine Downloaded from nejm.org on September 28, 2012. For personal use only. No other uses without permission. Copyright 2011 Massachusetts Medical Society. All rights reserved.