**5. Critical thinking on induced pluripotency**

Although iPSCs are highly similar to ESCs in biology and function, an increasing body of literature describes defects and subtle differences between the two pluripotent cell types. Effective characterization of these phenomena is critical and will likely give rise to new and more stringent criteria by which reprogrammed cells can be evaluated for suitability.

One of the earliest characterizations of reprogrammed cells was the demonstration of DNA demethylation on the promoters of genes involved in pluripotency, such as Oct3/4 and Nanog (Okita et al., 2007; Takahashi and Yamanaka, 2006). Although these promoters exhibited near-total demethylation, indicating an activation of gene transcription, the demethylation was rarely as complete as the pattern observed in ESCs, and this was particularly true for early-passage (newly-created) iPSCs. Although these differences were small, genome-wide analysis of methylation patterns outside of these specific genes has revealed significant errors in epigenetic reprogramming (Lister et al., 2011). On a genomic scale, iPSC and ESC methylomes are similar, but most iPSC methylomes analyzed had megabase-sized loci of aberrant DNA methylation, which persist long after reprogramming and even after differentiation. Some of these loci are shared among distinct iPSC lines, suggesting that certain regions of the methylomes are susceptible to aberrant and incomplete reprogramming. There have also been reports of differences in gene expression patterns between ESCs and iPSCs, although a recent study found that most of these differences are laboratory-specific and can be attributed to microenvironment differences in growth conditions from one laboratory to the next (Newman and Cooper, 2010).

DNA sequence defects have also been described in iPSCs. Early-passage iPSCs display a range of polymorphism in copy number variant (CNV) regions compared to their parental fibroblasts (Hussein et al., 2011). As with DNA methylation, it was also found that CNVs occurred more commonly in "fragile regions" of the genome. As CNVs arise from damaged DNA improperly repaired by homologous recombination, this phenomenon suggests DNA damage and replicative stress in cells undergoing reprogramming. However, while earlypassage iPSCs contain significantly more CNVs, a vast majority of these mutations put the cells at a selective disadvantage. Mid- to late-passage iPSCs therefore lose CNVs and soon approach a genomic state highly similar to ESCs. Point mutations in specific genes have also been identified in iPSCs, however unlike CNVs these display a nonrandom pattern of enrichment, with a majority occurring in proto-oncogenes and tumor suppressors (Gore et al., 2011). Half of these mutations can be traced back to the parental fibroblasts, which harbor these mutations in low frequencies; however the other half most likely arise during reprogramming and, more importantly, the subsequent selection and propagation steps. Oncogenic mutations are generalized to give a selective advantage to pluripotent cells, and although an accumulation in oncogenic mutations has also been demonstrated in ESCs, this study still establishes the need for extensive genetic testing of iPSCs before they are to be used on a clinical scale.

## **6. Conclusions**

688 Biomedical Science, Engineering and Technology

epigenetic reprogramming, but so far no such experiments have generated induced pluripotent stem cells. In one such study, human 293T epithelial cells treated with T cell extracts, assume a phenotype similar to T-cells, and a neuronal-like phenotype when treated with neural precursor extracts (Hakelien et al., 2002). Numerous attempts have been made to induce pluripotency by using extracts from ESCs, ECs, iPSCs and oocytes (Taranger et al., 2005; Zhu et al., 2010), however none of these have succeeded in producing stable iPSC lines.

Although iPSCs are highly similar to ESCs in biology and function, an increasing body of literature describes defects and subtle differences between the two pluripotent cell types. Effective characterization of these phenomena is critical and will likely give rise to new and

One of the earliest characterizations of reprogrammed cells was the demonstration of DNA demethylation on the promoters of genes involved in pluripotency, such as Oct3/4 and Nanog (Okita et al., 2007; Takahashi and Yamanaka, 2006). Although these promoters exhibited near-total demethylation, indicating an activation of gene transcription, the demethylation was rarely as complete as the pattern observed in ESCs, and this was particularly true for early-passage (newly-created) iPSCs. Although these differences were small, genome-wide analysis of methylation patterns outside of these specific genes has revealed significant errors in epigenetic reprogramming (Lister et al., 2011). On a genomic scale, iPSC and ESC methylomes are similar, but most iPSC methylomes analyzed had megabase-sized loci of aberrant DNA methylation, which persist long after reprogramming and even after differentiation. Some of these loci are shared among distinct iPSC lines, suggesting that certain regions of the methylomes are susceptible to aberrant and incomplete reprogramming. There have also been reports of differences in gene expression patterns between ESCs and iPSCs, although a recent study found that most of these differences are laboratory-specific and can be attributed to microenvironment differences in

more stringent criteria by which reprogrammed cells can be evaluated for suitability.

growth conditions from one laboratory to the next (Newman and Cooper, 2010).

DNA sequence defects have also been described in iPSCs. Early-passage iPSCs display a range of polymorphism in copy number variant (CNV) regions compared to their parental fibroblasts (Hussein et al., 2011). As with DNA methylation, it was also found that CNVs occurred more commonly in "fragile regions" of the genome. As CNVs arise from damaged DNA improperly repaired by homologous recombination, this phenomenon suggests DNA damage and replicative stress in cells undergoing reprogramming. However, while earlypassage iPSCs contain significantly more CNVs, a vast majority of these mutations put the cells at a selective disadvantage. Mid- to late-passage iPSCs therefore lose CNVs and soon approach a genomic state highly similar to ESCs. Point mutations in specific genes have also been identified in iPSCs, however unlike CNVs these display a nonrandom pattern of enrichment, with a majority occurring in proto-oncogenes and tumor suppressors (Gore et al., 2011). Half of these mutations can be traced back to the parental fibroblasts, which harbor these mutations in low frequencies; however the other half most likely arise during reprogramming and, more importantly, the subsequent selection and propagation steps. Oncogenic mutations are generalized to give a selective advantage to pluripotent cells, and although an accumulation in oncogenic mutations has also been demonstrated in ESCs, this study still establishes the need for extensive genetic testing of iPSCs before they are to be

**5. Critical thinking on induced pluripotency** 

used on a clinical scale.

Although induced pluripotency as a reprogramming technique currently brings significant concerns about carcinogenicity as well as genomic and epigenomic integrity, a significant portion of the ESC research community has jumped ship in recent years in order to study iPSCs. This is because of the exciting promise these cells hold, as well as the mainstream belief that the obstacles that come with them will be overcome. With applications in a variety of fields including regenerative therapies, disease modeling, animal cloning and genetic engineering, induced pluripotency is actively transforming the stem cell community. Given how young the field is, induced pluripotency has a surprisingly well-developed body of basic research, which has already contributed enormously to our understanding on developmental biology and epigenetics, as well as given us insights on a large number of modeled genetic diseases. Taken together, the current body of literature on induced pluripotency describes why it is a very exciting time to be a part of this field.

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