Human induced pluripotent stem cells (hiPSCs) represent a promising therapeutic tool for many diseases, and might be useful for regenerating aged tissues and organs at high risk of failure [1, 2]. However, the intrinsic self-renewal and pluripotency of hiPSCs potentially make them tumorigenic, hindering their clinical application [3–5]. hiPSCs are generated through epigenetic reprogramming of somatic tissue. It was initially thought that hiPSCs and human embryonic stem cells (hESCs) shared a high degree of epigenetic similarity [6, 7]. However, recent reports have indicated that substantial differences exist between hiPSCs and hESCs with regard to gene expression, miRNA expression and DNA methylation [8–10]. Cell-of–origin-specific genetic and epigenetic differences exist in hiPSCs  and some of these stem cell lines spontaneously differentiate during serial passage . Extensive evaluation of hiPSCs is consequently an essential component of the process required for their safe use in regenerative medicine.
Many types of malignant tumors are characterized by complex genetic and epigenetic alterations, including loss of heterozygosity (LOH) and loss of imprinting (LOI) [13, 14]. Such alterations are presumed to represent the second hit, according to Knudson’s two-hit hypothesis (OMIM #167000) . However, alterations in DNA methylation can also occur as the first hit during human carcinogenesis . Alterations in the expression of imprinted genes represent one of the most common changes seen in cancer [17, 18]. Some imprinted genes, including H19, GTL2, PEG1, PEG3, LIT1 (KCNQ1OT1)  and ZAC are known to act, or are strongly implicated to act, as tumor suppressor genes (TSGs). Furthermore, imprinted genes play key roles in regulating growth and differentiation . Thus the aberrant expression of imprinted genes may contribute to tumorigenesis or alter the differentiation potential of stem cells.
The monoallelic expression of imprinted genes is reliant on epigenetic mechanisms, most notably DNA methylation, which is established in the male and female germlines at discrete locations termed germline or gametic differentially methylated regions (gDMRs) . Imprinted domains generally contain several genes displaying allele-specific expression and gDMRs within these domains act as imprinting centers or imprint control regions for the domain . The majority of imprinted genes reside within these complex domains . Although gametic DMRs are maintained throughout the life of the organism, genes within the domain can be imprinted in tissue- and developmentally specific manners .
In a recent paper, we demonstrated that hiPSCs exhibit epigenetic patterns distinct from hESCs . After continuous passaging of the hiPSCs, these differences diminished such that over time the hiPSCs more closely resembled hESCs. However, we found that the imprinted DMRs showing abnormal methylation in early passage hiPSCs did not resolve during passaging. In this study we focused on the expression of imprinted genes in hiPSCs. Several reports on imprinted gene expression in hESCs demonstrate a substantial degree of instability . Less is known regarding the stability of imprints in hiPSCs, although some work has begun . We are particularly concerned with the stability of imprints in pluripotent stem cells during prolonged culture. Here, we examined the imprinting status and expression levels of eight imprinted genes and the methylation status of their DMRs in five independently derived hiPSCs. We found that the frequency LOI was very low in the early passaged lines. We also found that, in contrast, the epigenetic changes that took place at non-imprinted loci during prolonged culture for both normal and aberrant imprints were stably inherited despite prolonged passaging of the lines.