GENETIC AND EPIGENETIC INSTABILITY IN HUMAN INDUCED PLURIPOTENT STEM CELLS (hiPSC)

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Genetic and Epigenetic Instability in Human Induced Pluripotent Stem Cells (hiPSC)

Genetics and epigenetics variation and instabilities have been reported by several high-resolution genomic studies in the recent past irregularly to accumulate in human pluripotent stem cells. Detailed characterization of these changes in the proliferation, self-renewal and mainly on the developmental and malignant potential of these cells is essential for the understanding alteration the impact. The characterization is important for the enhanced and safe use of pluripotent cells for therapeutic use, such as regenerative cellular therapies utilizing differentiated derivatives of pluripotent cells. In this review, epigenetic and genomic stability of human pluripotent cells and the implications for stem cell research will be summarized.

An extensive study on genome stability for the human pluripotent stem cells and their derivatives, utilizing a combination of high-resolution genomic methods has been carried out. The structural genomic aberrations have been carried out by combining multiplex fluorescence in an in situ hybridization (M-FISH) and array comparative genomic hybridization (aCGH) techniques. Fascinatingly, different chromosomal and subchromosomal genomics have been identified in separate research and described amongst pluripotent stem cells and their derivatives. Some of the acquired modifications in the copy number variation are presumably acquired during the differentiation process and manipulation procedures while some of the other variations observed are probably inherited from the original cell lines. The need to study both the pluripotent stem cells and their differentiated stem cells using various methods arose from the previous findings, so as to evaluate and a certain their stability before using them in clinical therapies.

The necessity to develop therapies has increased the improvements in the differentiation and culture methods, legal permissions, industrial-scale cell culture and considerable investment from both private and public entities. However, as the concerns about the safety of hPSC therapies remain a major obstacle the production continuous. In other incidences, the transformed cells may harm the patient by inducing cancer or becoming cancerous. Two major concerns about tumorigenicity arise when it comes to cell types that are derived from human pluripotent stem cells (hPSCs)( Gore et al., 2011).Firstly, mutations in transplanted cells are a concern; genomic mutations are associated with tumorigenicity development. Secondly, undifferentiated human pluripotent stem cells can give rise to germ cell tumors that are normally benign when transplanted into mice that are immune-deficient during various researches. Though there are various concerns about residual undifferentiated cells in transplanted (hPSC) populations because all the clinical applications that are carried out require that the cells to be differentiated first before the process can commence. A more thorough characterization of the pluripotent stem cells and their derivatives of neuronal progenitor cell lines research first described structural genomic alterations in the primate’s stem cells (Thomson and Marshall, 1998). Since then the various research on genomic anomalies that have preceded the research by Thomson and Marshall (1998) have been identified in almost every single type of pluripotent stem cells, in spite of the cell culture condition used, modifications or manipulations incurred during the process. By comparison, epigenetic modifications are sensitive and dynamic to environmental stress and signals as was described by Robert Feil & Mario F. Fraga (2012) in their review journal. However, genomic variations and instabilities lack enough information that could potentially be got from differentiated acquired pluripotent stem cell types.

If acquired pluripotent cell types would be used as model in basic research or cell therapy, it’s essential that their genomic integrity should be thoroughly characterized and analyzed extensively so as to avoid any potential danger that they might pose to a patient. The idea that tumorigenicity and cancerous variants could grow in a relatively shorter duration was dismissed after covering the completion of a typical differentiation procedure that took 1 to 3 weeks (Stephenson et al, 2010; Winkler et al, 2009). According to a separate research conducted by Tomazou et al., (2010) all human pluripotent stem cells and human embryonic stem cells hold a very big potential for clinical and therapeutic applications. This is because they hold differentiation capacities and very incredible self-renewal they hold that makes them a possible source for large-scale quantities of differentiated cells for therapeutic biomolecule production, drug screening, cell therapy and toxicological studies and experiments.

In a highly replicated study for the identification of passing effects that might rise from various methods used and differential substrate constituents taking longer time in culture on epigenetic and genetic instabilities and phenotypic characterization of human pluripotent stem cells was achieved by Biancotti et al (2010). In both the research on human-induced pluripotent stem cells and human pluripotent stem cell researches. They observed that various accumulation in genetic changes and enzymatic passaging on a feeder free substrate was directly proportional to the changes that are seen in mechanical passaging on feeder layers. They observed in their study that extensive culture of human embryonic stem cells over a long duration of time under the conditions mentioned above indicated that even when used in conditions with the lowest overall rate of genetic alterations the number of changes rose drastically after going through 80 passages (which breaks down to a total number of 80 + 37=117). Another more detailed study on human embryonic stem cells indicated that the enzymatic passaging had minimal, or no effects at all on the buildup of genetic substrates compared to aberrations (Winkler et al., 2009).

 

Various methods for creating pluripotent stem cells by reprogramming for somatic cells from other tissues and species by the ectopic expression of distinct factors

 

Application Species of choice Donor cell type Reprogramming cocktail Delivery mode Recommendations
Drug screening and disease modeling. Human/pig Reprogrammable cells easily available from patients or cell repository OSK/OSKM; OSNL Retrovirus; RNA? The starting cell population may be limited, so competent methods are needed to generate models. Safety is not an important issue but avoiding integration would decrease genetic heterogeneity among cell lines
Study reprogramming mechanism Mouse Cells from chimeric mice from iPSCs obtained using an inducible system. OSK/OSKM* as reference, any additional factor possible Inducible lentivirus To understand reprogramming, by comparison of as many factors and cell types as possible
Cell therapy Human Reprogrammable cells readily available from cell repository, patients, or HLA-matched iPSCs obtained from cord blood Need to avoid inhibitors of tumor suppressor or potent oncogenes. Non-integrative Non-integrative
Research on pluripotency/differentiation Human/ Mouse mouse embryonic fibroblasts (MEFs)/fibroblasts *OSKM/OSK; OSNL‡ Retrovirus; RNA? To improve differentiation protocols and understand pluripotency, reproducible and reliable reprogramming methods are amongst best. Non-integrative methods may reduce genetic heterogeneity among cell lines

 

Subject to specific purpose of reprogramming, many methods have been developed when considering how to generate induced pluripotent stem cells (iPSCs); human leukocyte antigen (HLA); mouse embryonic fibroblasts (MEFs).

Shinya Yamanaka developed the * OSKM and *OSK combinations. In which *OSK demonstrates the combination of various transcription factors (TFs) SOX2, OCT4 and KLF4, and OSKM describes the combination of KLF4, OCT4, SOX2 and MYC.

James Thomson developed the ‡ OSNL combination. In which OSNL describes the combination transcription factors (TFs) OCT4, NANOG, LIN28 and SOX2, NANOG also known as Thomson factors.

 

 

Genetic instability

Karyotypic abnormalities such as trisomy of the chromosome had begun emerging in human embryonic stem cells begun emerging in early 2000. Some of this abnormities were reported by Hardarson et al. (2003). Later on other abnormalities were discovered by Laurent et al (2013) in their research; sub-chromosomal aberrations to whole chromosome aneuploidy, including point mutation are just some genome abnormalities that were indicated in their findings on their researched in human pluripotent stem cells studies. These findings were also supported with independent research carried out by Woltjen et al. (2009). Some duplication were also observed to be recurrent in human pluripotent stem cell studies (Woltjen et al. 2009)

 

Does human induce pluripotent stem cell reprogramming influence genetic stability?

According to Takahashi et al. (2007) in their research the first-generation of human induce pluripotent stem cell (hiPSC) lines were reprogrammed from somatic cells using lentiviral and retroviral vectors, which randomly integrate into the genome of the parent cell. Several other footprint free protocols have been developed such as those hypotheses by several separate research (Kim et al. 2009; Woltjen et al.,2009; Waren et al., 2011; Miyoshi et al.,2011) to minimize the risk of harmful re-activation and mutations of transgenes after reprogramming. However, there is no difference in the frequency and type of karyotypic changes described by Woltjen et al. (2009) or in the case of the mutation capacity explained in 34 independent researches were noticed in a comparison of non-integrative and integrative reprogramming methods (Waren et al.,2010). They proposed the method of reprogramming does not affect the genomic stability in human induced pluripotent stem cells (Woltjen et al. 2009). However, emphasis should be given to rare insertion mutagenesis events that occur in a culture of human pluripotent stem cells conditions, which are not readily detectable by the reprogramming methods. This additional emphasis could be relevant to human pluripotent stem cell (hiPSC) safety while supporting the use of non-integrating methods.

However evidence sufficient to support the reprogramming process, irrespective of the methodology used can induce genomic changes in hPSC (Hussein et al., 2011; Laurent et al.,2011). In their research they reported that the number of mutations and copy number variations (CNVs) was higher in human induced pluripotent stem cells (hiPSC) as compared to the corresponding fibroblast from which the human induced pluripotent stem cells were derived. Hussein et al.,(2011) revealed that on average, five protein-coding point mutations in the exomes of human induced pluripotent stem cells (hiPSCs) from exome sequencing, of which 50% occurred during or after reprogramming.

In the most recent studies, exome sequencing has shown that 74% of the mutations that were detected in human induced pluripotent stem cells were generated during reprogramming and 7% were caused by in vitro maintenance, whereas 19% were found to be in existence already in the parental fibroblast this is according to Laurent et al. (2011). However, a more recent study by Varela et al. (2012) showed that mouse induced pluripotent stem cells (miPSC) attributed this change in mutation frequency while reprogramming to the selection of the already existing sub-populations of mutant parental fibroblast. Both these two research indicated that reprogramming select for or cause for mutations and is subsequently the major obstacle for the genomic stability of human pluripotent stem cells (hiPSC).

Mechanism by which sensitivity leads to mutation in Human induced pluripotent stem cells (hiPSCs)

Chromosomal aberrations had been earlier been reported in hiPSC in which extensive study of 40 human embryonic stem cells (hEPSC) lines from which 1163 karyotypes were studied. These studies conclude that 12.9% of the human embryonic stem cells showed chromosomal aberrations (Taapken et al., 2011). From this finding it is essential to note that this frequency could still increase if more sensitivity methods were used; such as microarray as illustrated in other research involving various researchers (Maitra et al., 2005; Laurent et al., 2011; Autio et al., 2010). These changes in genetic aberrations could be attributed to numerous factors that could be influenced by the type of culture conditions and the period of culture.one of such factors involves enzymatic passaging that was identified to be a potential for clonal selection of aneuploidy clones; nevertheless, this hypothesis hasn’t been formally proven (Maitra et al., 2005; Mitalipova et al., 2005). However recently it was observed that karyotype abnormities in human embryonic stem cells can occur quickly within 10 passages and sub-karyotype abnormalities even much more rapidly when using single cell dissociation passaging. These findings in embryonic stem cells indicate that the culture conditions used for cell expansion and reprogramming are a critical factors for DNA stability during induced pluripotent stem cells generation. In recent studies many genomics alterations induced pluripotent stem cells were reliant on DNA extracted from cell before they undergo the 20th passage (Gore et al., 2011) from these numerous findings in the pluripotent stem cells have undergone numerous issues that require attention to prevent these deleterious mutational events, in specific: (1) to understand the means by which the generation of genomic alterations in hiPSC and (2) to identify the ideal culture conditions for pluripotent stem cell. Consequently, a qualitative assay that is necessary to gauge the genomic integrity of pluripotent stem cells to compare the impact on diverse parameters on the frequency of mutational events occurrences.

In normal circumstances, induced pluripotent stem cells can precisely repair a double-strand break in a DNA molecule majorly through the homologous recombination pathways (Johnson & Jasin., 2001). However, damage in error prone somatic cells that regularly repair their DNA damage through non-homologous end joining pathway (Lieber, 2007). The variance observed in this repair mechanism can explain the lower basal mutation rate in 3FB4-1 which the gets compared to the mouse embryonic fibroblast cells. Though 3FB4-1 displayed higher sensitivity to damage than the mouse embryonic fibroblast cells, when double strand breaks were introduced by ionizing radiation that might be one of the essential indicators of genomic instability for induced pluripotent stem cells. Therefore, it is most likely that genomic instability of induced pluripotent stem cells can be enhanced by regulation of DNA damage repair response to reduce the danger of tumor formation before use in clinical therapy procedures. Further research into the mechanism of DNA damage is very important to identify the underlying mechanisms in sensitivity of human induced pluripotent stem cells by DNA damage which could be approached by matching the repair capabilities of more cell types including embryonic stem cells, induced pluripotent stem cells and any other somatic cells or their derivatives.

 

Alterations can occur before and during in vitro maintenance.

Due to a restricted number of cells obtainable at the moment that human embryonic stem cell (hESC) lines are derived, it has not been conceivable to study in detail the extent to which the epigenomic or genomic changes mentioned above might already be present at the blastocyst stage. In the research conducted by Hardarson et al.,(2003) found that most excess in fertilized human blastocyst have been illustrated to be associated with chromosomal aberrations. It is also observed that single cell copy number variation analysis of 3 to 4 days old embryos that are in normal perfect conditions indicated chromosomal instabilities, uniparental disomic and mosaics as indicated by Martins-Taylor et al., (2012) point mutations and copy number variations are generated during reprogramming, and early passage human induced pluripotent stem cells consist of a mixed population of cells. Remarkably long term culture negatively selects for deletions or positively selects for amplifications in the human pluripotent stem cells as illustrated by Gore et al., (2011). This phenomenon had earlier been explained by Hussein et al., (2010) by showing that early passage in human induced pluripotent stem cells that are genetically mosaic and that proliferation of cells in culture that would rapidly select against mutated cell types. Recently the observation in the earlier two researches by Hussein et al., (2010) and Gore et al., (2011) were also observed by Martin-Taylor et al., (2012) that a class of copy number variations occurring at lower passages were lost at high passages, independently supporting the mosaic theory.

 

Genomic Changes Occur during Differentiation of Human Pluripotent Stem Cells (hPSCs)

As well as the genomic alterations that take place while human pluripotent stem cells are cultured in their pluripotent nature, it is also possible for such changes to occur during their differentiation. As culture conditions are changed to drive differentiation, new selective pressures are applied to the cells, possibly selecting for new genomic variants (Varela et al., 2011). This type of change is difficult to detect because differentiation is frequently associated with standard karyotyping procedures require dividing cells and decreases in proliferation. The earliest reported example of genomic changes arising during differentiation was found during a large-scale single nuclear polymorphism and genotyping study by Laurent et al.(2011). This research showed that an abnormal genomic subpopulation present in cultures of undifferentiated WA07 human embryonic stem cells was selected for in a cardiac differentiation experiment. In this example, after only 5 days, the differentiated population was revealed to be greatly increased for cells with many duplications in chromosome 20 (Irizarry et al., 2009).

Varela et al. (2012), however, indicated that when the NSCs with the 1q amplification had been injected into immune-compromised rat brains, they did not form tumors. Gains of chromosomes 17q and 12 have been reported to occur in human embryonic stem cells (hESC) lines as described by Draper (2004). He made it clear that the karyotype abnormalities frequently accumulate in human embryonic stem cells (hESCs) during in vitro maintenance. Comparison of the karyotype abnormalities that have been detected by Baker et al., (2007) revealed that the most common changes that amassed in human embryonic stem cells (hESCs) gains in chromosomes 12, 17 and, to a lesser extent, X chromosome (Irizarry et al.,2009) . These variations are also frequent in germ cell tumors. There are still little examples to know how often differentiation-driven selection occurs, and the functional consequences remain to be investigated. Though, it is essential to note that even when the undifferentiated human pluripotent stem cell population is independent of detectable genomic abnormalities, selection during differentiation can increase abnormal cells. Because these cells are the ones chosen for cell therapy, it is important that the final step before transplantation be a last check of the genomic state of the cells.

 

Karyotype Focal alterations.

In relation to the low-resolution karyotypic analyses, research studies by Taapken et al.,(2011) on pluripotent cells have provided more understandings into the genes and genomic regions that might be involved in the initiation for the enrichment of the experimentally determined karyotypic abnormalities in pluripotent stem cell populations. Numerous array comparative genomic hybridization (aCGH) studies have been undertaken on human pluripotent cells. Loss of heterozygosity changes occurrences in prolonged culture and copy number variants (CNVs) is to be blamed for the karyotype abnormities (Gore et al., 2011). Copy number variations spanning genes that have been associated with pluripotency which were reported by Mayshar et al., (2010), which occurs very close to NANOG, have been identified in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). This discovery is that NANOGP1 is the only NANOG pseudogene that is expressed in human embryonic stem cells, and many pseudo genes may function as competing targets for miRNAs, thus affecting translation of the transcripts from the analogous functional gene. Remarkably, NANOGP1 and NANOG are located on chromosome 12 ( Irizarry et al.,2009), which is the most frequently observed hotspot for genomic alterations in both human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). However, according to Werbowetski-Ogilvie et al.(2009) variation in the genomic region spanning NANOGP1 is frequently found in human populations, demonstrating that this alteration is not specifically augmented in hESCs

In studies carried out by Baker et al., (2007) analyzing the genetic instability in pluripotent stem cell lines, was important as it addressed whether the observed variation among cell lines from different individuals is ahead and differs from the normal individual to individual variation that exists in human populations, as demonstrated by the NANOGP1 locus. Fascinatingly, copy number variants seen among individuals in somatic human tissues include a roughly equal distribution of amplifications and deletions, whereas in human embryonic stem cells almost 75% of the genomic areas covered by copy number variants (CNVs) are amplifications. Likewise, the average length of three copy duplications has been reported to be lower in non-pluripotent cells than in human induced pluripotent stem cell and human embryonic stem cells. Furthermore, somatic cells and human embryonic stem cells, human embryonic stem cells have been described to carry a significantly larger copy number variation than somatic cells.

For dependable analyzed data, it is important to use an appropriate reference genome, from sequencing studies or array-comparative genomic hybridization (aCGH) which at times is affected by the genome or reference genomes used to normalize the kind of data (Beroukhim et al., 2010). For instance, human induced pluripotent stem cells (hiPSC) data may be normalized against reference genomes from parental fibroblasts, human embryonic stem cells (hESCs), karyotypically normal human induced pluripotent stem cells (hiPSC) or HapMap data (International HapMap Consortium., 2003). When comparison of different cell types is carried out, it is vital that the data be normalized in a similar way. Modern platforms facilitate reference genomes to be constructed from multiple sources and samples this is according to International HapMap Consortium (2003), whereas the results obtained using early dual colour based on array-comparative genomic hybridization (aCGH) platforms were based on matching the test sample to a single reference genome sample.

 

 

 

 

Epigenetic Instability

The hESC lines are derived from blastocysts during an epigenetically sensitive and dynamic stage of development. During early developmental stage that involves major epigenomic reprogramming of the chromatin, including histone tail modifications and changes in both DNA methylation, which contribute to gene regulation takes place. During gametogenesis, epigenetic patterns are re-established, and gametic imprints are placed to facilitate a subset of genes to be mono-allelically expressed according to the parental origin of the allele. Many of these imprinted genes are resilient to the subsequent global demethylation that occurs between blastocyst formation and fertilization. In the female embryos, the inactivation of one of the pairs X chromosomes starts at the eight cell stage and is completed by the blastocyst stage. Derivation of Human Embryonic Stem cells (hESC) lines from blastocysts during this epigenetically sensitive and unstable phase and further expansion in non-physiological conditions under continuous mitogenic stress is probably to introduce alterations in the epigenome of the cell lines (Baker et al., 2007) .

In the same way, reprogramming human induced pluripotent stem cells (hiPSCs) from the somatic tissue can induce epigenetic anomalies in the cells or can be incomplete. Epigenetic regulation is crucial for normal aging and development this according to Smith (2011). In her research, she suggests that any disturbances in epigenetic regulation may alter the developmental potential of the cells and may predispose to malignancies or diseases. Therefore, cautious characterization of individual pluripotent cell lines is important for monitoring and defining the quality of the cells. Several other studies have also been carried out to support her epigenetic stability analysis of the pluripotent cells

Pluripotent cells epigenome is extremely sensitive and dynamic to variation. It is now known that instability and variation in DNA methylation of a subset of developmental and imprinted genes is a characteristic for cancer cells and pluripotent cells, and these alterations may provide growth advantages for both cell types (Anastasia et al.,1992) . The section of the genes affected varies in different studies, and common changes have only been found in a few genes. But, most of the research carried so far has analyzed a limited number of pre-selected genes. More comprehensive, high-resolution studies that use impartial sequencing-based methods are still limited in number. Additionally, the stability of histone modification patterns during extended culture has not been reported in any of the previous studies. Comprehensive large-scale genome-wide studies controlling the important technical variables, such as time and conditions in culture, are needed to describe the level of normal epigenetic variation in pluripotent cells and the changes happening during in vitro maintenance that have biological consequences.

 

 

Relationship between Genomic and Epigenetics Instability in Human Pluripotent Stem Cells (hPSCs) and Tumorigenesis/ Tumor Cells

Earlier studies indicated that the teratoma-forming tendency of induced pluripotent stem cells (iPSCs) is variable; nevertheless, the relationship between genomic instability and tumorigenic potential in human induced pluripotent stem cells (hiPSCs) remains to be totally explained. In, this review we evaluated the malignant potential of human induced pluripotent stem cells from some of the research data available.

 

Cancer Associated Genetic Alterations in Cultured Pluripotent Cells.

Human embryonic stem cells (hESCs) with karyotypic abnormalities share physiognomies with cancer cells and may become malignant after transplant into mice this is according to research conducted by Werbowetski-Ogilvie et al.(2009). Therefore, it is possible that the genomic changes seen in pluripotent cells provide selective growth advantages related to those observed in malignant cells arising in somatic tissues. The common amplification hotspot in pluripotent cells at the 20q, 11 loci has also been associated with cancers according to Beroukhim et al. (2010). The trisomy of chromosome 12, now recognized as the most recurrent genetic alteration in pluripotent stem cells, is also used as an indicative marker for germ cell tumors and it is amongst the most frequent alterations witnessed in chronic lymphocytic leukaemia according to studies by Anastasia et al.,(1992). Moreover, as mentioned above, amplification of MYC can occur in cancers and can also in human embryonic stem cells (hESCs).

Cancer Associated with Epigenetic Modifications in Cultured Pluripotent Cells.

There are various similarities between epigenetic alterations in cancer cells and pluripotent cells: both these cell types have been shown to indicate presence of instability in imprinting, XCI or DNA methylation, which has been identified to frequently affects the same areas as with the case with both cell types (Werbowetski-Ogilvie et al., 2009; Gore et al., 2011). A research of culture induced DNA methylation modifications in 14 cancer-associated gene promoters in their late and early passages in 9 human embryonic stem cells (hESC) lines found methylation alterations in three of these promoters, most commonly involving the tumor suppressor gene RASSF1 ( Irizarry et al.,2009). The RASSF1 protein is associated in the regulation of crucial cellular functions, such as cell cycle progression and apoptosis. Also, methylation of the RASSF1 promoter is an early indicator for the progression of tumorigenesis. Irizarry et al.(2009) also reported in their research that genes that are differentially methylated in somatic cells and in pluripotent cells in a tissue-specific manner overlap with the genes that are differentially methylated in Adenocarcinomas, and these differences affect CpG island. Other studies that support the arguments found that genes that are hyper-methylated in adult cancers had a bivalent chromatin state in embryonal carcinoma cells and human embryonic stem cells (hESCs) (Anastasia et al., 1992).

 

 

Conclusions and Future of Human Pluripotent Stem Cell (hPSC) Research  

In the light of the most recent research, it is now clearly understood that genomic modifications buildup in the pluripotent stem cells and these changes can occur at various levels during in vitro maintenance and culture. Hence, it is not conceivable to clearly outline the passage number thresholds to guarantee safety. Though, the risk of abnormalities rise is evident in relatively prolonged culture durations. Hence, the early passages are less probable to contain genomic modifications and alterations. However, most of the pluripotent stem cell lines can maintain the normal karyotype, even during longer in vitro maintenances of culture, therefore giving a valuable resource for medical and therapeutic applications. The reason for which some of the recurrent abnormalities are different between human induced pluripotent stem cells and human induce pluripotent stem cells and human embryonic stem cells it is fascinating and requires further investigations.

There is strong evidence indicating that pluripotent cells and some of their differentiated derivatives bearing genomic abnormalities can show malignant features. These findings further stress the need for careful screening before considering the use of these cells in therapies and therapeutics. While the genetic stability of stem cells can be a serious safety concern for regenerative medicinal applications, these stem cell lines carrying specified genetic alterations and those derived from inner cell mass of blastocysts, such as embryonically lethal mutations, have continually provided valuable tools and protocols for research aimed at the chromosomal aneuploidies and model genetic abnormalities as discussed by Biancotti et al., (2010). These cell lines should be well preserved and made available to stem cell community for further studies and research instead of discarding the lines.

The epigenome of pluripotent cells is highly sensitive and dynamic to variation. We now understand that instability and variation in DNA and XCL methylation of a subset of developmental and imprinted genes are characteristic for cancer cells and pluripotent cells, and these alterations may provide growth advantages for both the cell types of embryonic stem cells and human pluripotent stem cells. These groups of genes affected by various aberrations differs in different studies, and frequent changes have only been associated with a few of these genes. Though, most of the studies undertaken so far have analyzed a restricted number of pre-selected genes. Comprehensive, high-resolution studies that use unbiased sequencing-based methods described by Tomazou et al., (2010) are still limited in number. Moreover, to our knowledge, the stability of histone modification patterns during extended culture has not been reported.

Even though this Review focuses on pluripotent cells, it is significant to note that regenerative medicine applications will naturally first involve the in vitro differentiation of these pluripotent cells into chosen tissue types. Consequently, to maximize safety, it will be important to monitor the final differentiated cell products and to characterize any additional changes that may occur during differentiation and culture process. These characterizations have not yet been extensively conducted by researchers, but the importance of such observations is underlined by recent discoveries of recurrent instability of chromosome 1q and rapidly rising genomic aberrations after directed differentiations in the culture of neural derivatives that were differentiated from pluripotent cells. Not all mutations are harmful, and insights from cancer research are likely to help to describe which mutations in pluripotent cells that are of the most relevant for safety. Hence, monitoring pluripotent cells may ultimately involve screening for specific rather than all alterations. However, there is not such a thing as a perfect human genome that carries no risk there is only acceptable risk.

References

 

 

Autio, R., Rahkonen, N., Kong, L., Harrison, N., Kitsberg, D., Borghese, L., . . . Lahesmaa, R. (2010). High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity. Nature Biotechnology.

Baker, D. E., Harrison, N. J., Maltby, E., Smith, K., Moore, H. D., Shaw, P. J., . . . Andrews, P. W. (2007). Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nature Biotechnology.

Doi, A., Park, I., Wen, B., Murakami, P., Aryee, M. J., Irizarry, R., . . . Feinberg, A. P. (2009). Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nature Genetics.

Draper, J., Smith, K., Gokhale, P., Moore, H., Maltby, E., John-son, J., . . . Andrews, P. (2004). Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.

Fraga, M. F., & Feil, R. (2012). Epigenetics and the environment: emerging patterns and implications. Nature Reviews Genetics.

Gibbs, R. A., Belmont, J. W., Hardenbol, P., Willis, T. D., Yu, F., Yang, H., . . . Waye, M. M. (2003). The International HapMap Project. Nature.

Gore, A., Li, Z., Fung, H., Young, J. E., Agarwal, S., Antosiewicz-Bourget, J., . . . Zhang, K. (2011). Somatic coding mutations in human induced pluripotent stem cells. Nature.

Hussein, S. M., Batada, N. N., Vuoristo, S., Ching, R. W., Autio, R., Närvä, E., . . . Otonkoski, T. (2011). Copy number variation and selection during reprogramming to pluripotency. Nature.

Israel, M. A., Yuan, S. H., Bardy, C., Reyna, S. M., Mu, Y., Herrera, C., . . . Goldstein, L. S. (2012). Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells. Nature.

Johnson, R. D., & Jasin, M. (2001). Double-strand-break-induced homologous recombination in mammalian cells. Biochemical Society Transactions.

Kim, D., Kim, C., Moon, J., Chung, Y., Chang, M., Han, B., . . . Kim, K. (2009). Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins. Cell Stem Cell.

Laurent, L. C., Ulitsky, I., Slavin, I., Tran, H., Schork, A., Morey, R., . . . Loring, J. F. (2011). Dynamic Changes in the Copy Number of Pluripotency and Cell Proliferation Genes in Human ESCs and iPSCs during Reprogramming and Time in Culture. Cell Stem Cell.

Lieber, M. R. (2007). The Mechanism of Human Non-homologous DNA End Joining. Journal of Biological Chemistry.

Maitra, A., Arking, D. E., Shivapurkar, N., Ikeda, M., Stastny, V., Kassauei, K., . . . Chakravarti, A. (2005). Genomic alterations in cultured human embryonic stem cells. Nature Genetics.

Mannion, J. D., Bitto, T., Hammond, R. L., Rubinstein, N. A., & Stephenson, L. W. (2010). Histochemical and Fatigue Characteristics of Conditioned Canine Latissimus Dorsi Muscle.

Mayshar, Y., Ben-David, U., Lavon, N., Biancotti, J., Yakir, B., Clark, A. T., . . . Benvenisty, N. (2010). Identification and Classification of Chromosomal Aberrations in Human Induced Pluripotent Stem Cells. Cell Stem Cell.

Mitalipova, M. M., Rao, R. R., Hoyer, D. M., Johnson, J. A., Meisner, L. F., Jones, K. L., . . . Stice, S. L. (2005). Preserving the genetic integrity of human embryonic stem cells. Nature Biotechnology.

Miyoshi, N., Ishii, H., Nagano, H., Haraguchi, N., Kano, Y., Nishikawa, S., . . . Mori, M. (2011). Reprogramming of Mouse and Human Cells to Pluripotency Using Mature MicroRNAs. Cell Stem Cell.

Taapken, S. M., Nisler, B. S., Newton, M. A., Sampsell-Barron, T. L., Leonhard, K. A., McIntire, E. M., & Montgomery, K. D. (2011). Karyotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells. Nature Biotechnology.

Thomson, J. A., & Marshall, V. S. (1998). Primate embryonic stem cells.

Tomazou, E. M., Brinkman, A. B., Müller, F., Simmer, F., Gu, H., Jäger, N., . . . Meissner, A. (2010). Quantitative comparison of genome-wide DNA methylation mapping technologies. Nature Biotechnology.

Tsai, S., Hardison, N. E., James, A. H., Motsinger-Reif, A. A., Bischoff, S. R., Thames, B. H., & Piedrahita, J. A. (2011). Transcriptional profiling of human placentas from pregnancies complicated by preeclampsia reveals disregulation of sialic acid acetylesterase and immune signalling pathways. Placenta.

Warren, L., Manos, P. D., Ahfeldt, T., Loh, Y., Li, H., Lau, F., . . . Rossi, D. J. (2010). Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA. Cell Stem Cell.

Woltjen, K., Michael, I. P., Mohseni, P., Desai, R., Mileikovsky, M., Hämäläinen, R., . . . Nagy, A. (2009). piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature.