Characterization of the individual capacity for repair of genotoxic damage of a Bulgarian hESC line and two commonly used stabilized cell lines

Abstract The number of stem cell lines worldwide grows steadily, including disease-specific lines and lines derived from specific tissues. The proportion of stem cell lines that were established in strictly xeno-free conditions is rapidly increasing, but it is still unclear whether these lines may be maintained in vitro for prolonged periods of time with satisfactory survival rates and minimal loss of their ‘stemness’ properties. The efficiency of repair of DNA damage has recently emerged as an important factor for maintenance of stem cells in culture with minimal genomic changes and preservation of the undifferentiated state. In this study we investigated the individual capacity for repair of DNA damage/maintenance of genomic integrity and additional markers in one human embryonic stem cell (hESC) line derived in Bulgaria from a discarded embryo and two of the human T-leukaemia cell lines commonly used for research purposes (T-1301 and Jurkat E6-1). Knowledge about the status of the studied cancer cell lines may be valuable for research purposes. Data about the individual repair capacity and the genetic risk for common late-onset diseases of newly established hESC lines as well as hESC lines currently in use may become a valuable tool in the assessment of the applicability of pluripotent human cell lines for research purposes, clinical trials and, potentially, clinical applications.


Introduction
The number of stem cell lines worldwide continues to grow, including disease-specific lines and lines derived from specific tissues such as dental pulp, placenta, amniotic fluid and others [1][2][3]. Despite the worldwide effort to establish and maintain new cell lines exclusively in xeno-free conditions, many of the ones routinely used have been derived and maintained in conditions that included animal sera and other undefined components. This precludes their potential use in clinical applications [4][5][6]. The proportion of stem cell lines that were created in strictly xeno-free conditions currently increases, but it is still unclear whether all these lines may be maintained in vitro for prolonged periods of time with satisfactory survival rates and minimal loss of their 'stemness' properties. The efficiency of repair of DNA damage has recently emerged as an important factor for maintenance of stem cells in culture with minimal genomic changes and preservation of their 'stemness' characteristics [7,8]. This is especially valid for disease stem cell lines for diseases where the aetiology had been specifically linked with proliferation of cancer stem cells (e.g. leukemias) [9,10]. At the same time, it became clear that reprogramming of somatic cells to pluripotency was dependent on the cells' capacity for maintenance of the integrity of their genome [11,12] and that activation of the expression of specific protein factors of DNA repair was required for differentiation along specific cell lineages [13,14]. Thus, characterization of the individual capacity for repair of DNA damage/maintenance of genomic integrity has recently begun to gain importance in stem cell research, especially in the light of potential clinical applications [4,6,15]. There is significant variation in the capacity for management of genotoxic damage among clinically healthy individuals. These differences are partly conferred by carriership of variant alleles of genes coding for key proteins of DNA repair and maintenance of genomic integrity (assessable by genetic analysis) and partly by interaction of the genetic background of the individual with environmental and lifestyle factors (assessable by more complex methodology) [16,17]. Typically, carriership of variant alleles of genes of DNA repair does not matter significantly in young and healthy individuals. Nevertheless, its influence may become significant in the course of ageing and in conditions of increased genotoxic stress (e.g. specific diseases and conditions such as insulin-resistant phenotypes but also in individuals undergoing genotoxic treatments) [16,18]. As variance in genes coding for DNA repair is very common, it is likely that the majority of the presently available human stem cell lines carry subtle deficiencies of DNA repair/maintenance of genomic integrity that may affect their survival in vitro after repeated passaging and may increase the risk for carcinogenic transformation.
At present, there is only a limited number of studies about the role of the variance of the individual repair capacity in the maintenance in vitro of stem cell lines. The individual capacity for DNA repair of the majority of the commonly used cell lines is also under-characterized despite the fact that they are routinely used for control purposes.
With these considerations in mind, we studied the individual capacity for repair of DNA damage/maintenance of genomic integrity in one hESC line (codenamed BABE1) derived in Bulgaria from an embryo left over after an assisted reproduction cycle [19] and two of the commonly used human T-cell leukaemia cell lines (T-1301, a derivation of the CCRF-CEM line, and Jurkat E6-1) [20,21]. The panel of markers for assessment of individual repair capacity comprised the polymorphisms ТР53 Pro72Arg (rs1042522, marker for DNA damage response/maintenance of genomic integrity); XPC ins83 (83 bp insertion/5 bp deletion in intron 9 of the ХРС gene, marker for global genomic repair by nucleotide excision repair (NER)); C8092A (rs3212986) in the 3′-UTR of the ERCC1 gene (marker for efficiency of NER); Asp312Asn (rs1799793) lys751gln (rs13181) in the XPD (ERCC2) gene (also marker for efficiency of NER); Thr241Met (rs861539) in the XRCC3 gene (marker for efficiency of repair of double strand breaks (DSBs)); Arg399gln (rs25487) in the XRCC1 gene (marker for efficiency of base excision repair, NER and single-strand break repair); and Ala222Val (C677T; rs1801133) in the MTHFR gene (marker for detoxification of genotoxic metabolites) [22][23][24][25][26].
Knowledge about the status of the studied T-cell leukaemia lines for these markers may be valuable exclusively for research purposes. Nevertheless, exhaustive characterization of the existing and newly derived human stem cell lines for their capacity to repair genomic damage and for carriership of common mutations and polymorphisms associated with predisposition to multifactorial disease and/or susceptibility to infectious agents may be useful for potential clinical applications of human stem cell preparations.

Ethics statement
Informed consent for the research use of human biological materials was obtained. The procedure was approved by the Ethical Committee of Sofia University 'St. Kliment Ohridski' .

Experimental procedures
Human ESCs were derived from a fresh embryo discarded after an assisted reproduction procedure and propagated in vitro as described in Arabadjiev et al. [19]. The embryo was donated for research purposes by the biological parents after informed consent. The procedure was endorsed by the Ethical Committee of Sofia University 'St. Kliment Ohridski' .
The in vitro stemness assay of the resulting cell line BABE1 was carried out by RT-PCR using RNA extracted from 10 5 to 106 cells with RNAeasy kit (qiagen) according to the instructions provided by the manufacturer. Purified RNA was subjected to treatment with RNase-free DNase I (Fermentas) to avoid misamplification of genomic sequences with high degree of similarity. Then, 3-5 µg of the DNase I-treated and purified RNA was used for first strand cDNA synthesis with RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas) and oligo-dT, according to the instructions provided by the manufacturer. OCT4, SOX2, NANOG and KLF4 were used as 'stemness' markers. The housekeeping genes GADPH and UBC were used as positive controls for RT-PCR. A volume of 1-2 µl of the first-strand reaction mix was used without purification for second chain synthesis and in vitro amplification (carried out in a single tube for 35-40 cycles). Primer design for second-chain synthesis and in vitro amplification was carried out using Vector NTI (Fisher Scientific) except for markers GADPH [27] and NANOG [28].
T-1301 and Jurkat E6-1 immortalized cell lines (human T-cell leukaemia) were purchased from Sigma-Aldrich and cultured in suspension in RPMI 1640 supplied with 2 mmol/l l-glutamine and 10% fetal bovine serum in 5% CO 2 at 37 °C, at density 3-9 × 10 5 cells/ml, according to the manufacturer's instructions.

Results and discussion
Sequences of primers used for second-chain synthesis and in vitro amplification in the 'stemness' assay of the hESC line B1 are listed in Table 1.
The stemness assay showed that the basic 'stemness' markers OCT4, SOX2, NANOG and KLF4 were expressed in the hESC line BABE1 but not in the control cell lines. The latter is unsurprising, as stemness markers may or may not be expressed in different types of T-cell leukaemia cells. KLF4 may be expressed in Jurkat E6-1 cells, albeit at a low level [29].
The results of the analysis of individual markers are presented in Table 2.
T-1301 and Jurkat E6-1 are pseudodiploid cell lines with modal chromosome number 46, carrying XX (for T-1301) and Xy genotypes (for Jurkat). Both lines carry mutated gene copies in the TP53 locus resulting in expression of mutant cancer-specific p53 [30,31].
Our study shows that T-1301 and Jurkat E6-1 carry the 72Arg allele of the TP53 Pro72Arg polymorphism, which is the preferred target for mutagenesis in some cancers [32].
T-1301 carries a single insertion allele in the XPC locus. The latter has been associated with subtly decreased capacity for recognition and repair of genotoxic damage in the untranscribed regions of the genome and increased risk for different types of cancer [33][34][35][36]. Carriership of the deletion allele of XPCins83 has been associated with low level of residual DNA damage after controlled treatments with ionizing radiation and other genotoxic agents [35,37]. Association with increased risk for leukaemia has not been reported so far, although all tissues with naturally rapid turnover (skin, mucosa, endothelial layer of blood vessels and haematopoietic tissue) are dependent on XPC-dependent detection and repair of damage in untranscribed genomic regions.
Jurkat E6-1 cell line carried a single C allele for the ERCC1 C8092A polymorphism, known to be associated with lower stability of the ERCC1 transcript, and, respectively, with lower levels of ERCC1 mRNA and protein [38]. Carriership of the C8092A polymorphism of ERCC1 gene is associated with increased risk for various types of cancer [39,40], although association with T-cell leukaemia has not been reported.
Jurkat E6-1 also carries a single gln allele by the XRCC1 Arg399gln polymorphism, associated with subtly decreased capacity for repair by base excision. The latter is associated with increased risk for cancer [41].
Both control cell lines carry a heterozygous Thr/Met genotype by the polymorphism XRCC3 Thr241Met. This polymorphism is associated with subtly decreased capacity for repair of DSBs in DNA by the mechanism of homologous recombination. Carriership of the Met allele at this locus has been shown to be a factor increasing the genomic instability in cultured cells [42] and, potentially, a factor in the risk for development of some types of cancer but not leukaemia [43][44][45]. genotypes at the other studied loci were wildtype, except for carriership of a single T allele at the MTHFR locus for T-1301. Heterozygous C677T carriership is very common in all populations [46]. There have been numerous reports in the specialized literature that 677 T alleles at the MTHFR locus may increase the risk for various cancers [47,48].
Some degree of genetic propensity for genomic instability is apparent in both T-1301 and Jurkat E6-1. To the best of our knowledge, up to the present moment there is no data about the carrier status of common polymorphisms in genes responsible for the capacity to detect and repair DNA damage even in the commonly used cancer lines. Such information may be useful for the purposes of research of genetic bases of cancer.
The BABE1 line carries a heterozygous Pro72Arg genotype at the TP53 Pro72Arg locus. This is a very common genotype in the Bulgarian population and is not associated with any significant effects on the phenotype [49].
The genotype of the BABE1 hESC line at the XPD, ERCC1 and XRCC1 loci was wildtype. The most common prothrombotic polymorphism (MTHFR C677T) was not present in the BABE1 human stem cell line.
The BABE1 hESC line carried a single insertion allele by the polymorphism XPCins83 and a single 241Met allele by the XRCC3 Thr241Met polymorphism. As was mentioned above, carriership of polymorphisms in the XPC gene was linked to increased risk for cancer. In the Bulgarian population, carriership of the heterozygous ins/del genotype has been shown to be associated with increased risk for cardiovascular disease [17,50]. Similarly, carriership of the variant allele at the XRCC3 Thr241Met locus confers increased risk for cancers [43][44][45].
Stem cell lines carrying polymorphisms in key DNA repair genes may be susceptible to loss of capacity for proliferation and differentiation during in vitro propagation. This may result in loss of the hESC line or carcinogenic transformation. Information about the capacity for identification and repair of genotoxic damage/management of genomic integrity may be used in the assessment of potential genomic stability of ESC lines and their applicability for research and potential clinical applications.
The number of hESC lines has grown significantly in the last decade. Nevertheless, the majority of the pluripotent lines currently in use may be unfit for applications apart from research, as they have not been derived and grown in xeno-free conditions and have been subjected to multiple passaging. Many authors already propose that the use of the most of the ESC lines that are currently used ought to be discontinued and new ESC lines must be established following the gMP guidelines [1][2][3]51]. Other authors propose that mutation rates must be routinely assessed in hESC lines and must be factored in the considerations of the potential applications of stem cell lines [15,52]. Analysis of the individual repair capacity of newly established hESC lines as well as hESC lines currently in use may aid in the assessment of their applicability for research purposes, clinical trials and, potentially, clinical applications.

Conclusions
Human cell lines commonly used in research are typically thoroughly characterized with regard to specific features of their biology, their safety for use in research and, in some cases, their immunological characteristics. Characterization of the genetic bases of capacity for DNA repair and maintenance of genomic integrity of human cell lines is still under-researched although available data show that it may reflect on the capacity for maintenance of cells in vitro and, in the case of stem cells, on their capacity for proliferation and differentiation. This article presents the results of locus-by-locus assessment of eight markers for individual capacity for repair of genotoxic damage in one human ESC line established in Bulgaria and two commonly used human T-leukaemia cell lines. Such data may be potentially useful in the assessment of applicability of human cell lines for research purposes, and, potentially, for clinical applications in the future.