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 [Citation1–3]. 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 [Citation4–6]. 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 [Citation7, Citation8]. 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) [Citation9, Citation10]. 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 [Citation11, Citation12] and that activation of the expression of specific protein factors of DNA repair was required for differentiation along specific cell lineages [Citation13, Citation14]. 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 [Citation4, Citation6, Citation15]. 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) [Citation16, Citation17]. 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) [Citation16, Citation18]. 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 [Citation19] 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) [Citation20, Citation21]. 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) [Citation22–26].
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.
Materials and methods
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. [Citation19]. 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 105 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 [Citation27] and NANOG [Citation28].
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% CO2 at 37 °C, at density 3–9 × 105 cells/mL, according to the manufacturer's instructions.
Assessment of the individual repair capacity of the BABE1 line and the control T-cell leukaemia cell lines was carried out using markers ТР53 Pro72Arg (rs1042522); XPC ins83; ERCC1 C8092A (rs3212986); XRCC3 Thr241Met (rs861539); XRCC1 Arg399Gln (rs25487); XPD (ERCC2) Asp312Asn and Lys751Gln (rs13181); MTHFR Ala222Val (rs1801133) in the MTHFR gene [Citation22–26].
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. Primer sequences used for second-chain synthesis and subsequent in vitro amplification for the purposes of ‘stemness’ assay for the human ESC line BABE1.
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 [Citation29].
The results of the analysis of individual markers are presented in .
Table 2. Locus-by-locus genotypes of the human stem cell line BABE1 and the two control human T-cell leukaemia cell lines.
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 [Citation30, Citation31].
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 [Citation32].
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 [Citation33–36]. 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 [Citation35, Citation37]. 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 [Citation38]. Carriership of the C8092A polymorphism of ERCC1 gene is associated with increased risk for various types of cancer [Citation39, Citation40], 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 [Citation41].
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 [Citation42] and, potentially, a factor in the risk for development of some types of cancer but not leukaemia [Citation43–45].
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 [Citation46]. There have been numerous reports in the specialized literature that 677 T alleles at the MTHFR locus may increase the risk for various cancers [Citation47, Citation48].
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 [Citation49].
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 [Citation17, Citation50]. Similarly, carriership of the variant allele at the XRCC3 Thr241Met locus confers increased risk for cancers [Citation43–45].
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 [Citation1–3, Citation51]. 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 [Citation15, Citation52]. 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.
| Abbreviations | ||
| ESC | = | embryonic stem cells |
| APOE | = | Apolipoprotein E |
| DSB | = | double-strand break |
| ERCC1 | = | excision repair cross-complementation group 1 |
| ERCC2 | = | excision repair cross-complementation group 2 |
| MTHFR | = | 5,10 -methylenetetrahydrofolate reductase |
| NER | = | nucleotide excision repair |
| PT | = | prothrombin |
| TP53 | = | tumour protein p53 |
| UTR | = | untranslated region |
| XPC | = | xeroderma pigmentosum complementation group C |
| XPD | = | xeroderma pigmentosum complementation group D |
| XRCC1 | = | X-ray repair cross-complementing protein 1 |
| XRCC3 | = | X-ray repair cross-complementing protein 3 |
Data availability statement
The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials.
Disclosure statement
The authors declare no conflict of interest.
Funding
The author(s) reported there is no funding associated with the work featured in this article.
References
- Kobold S, Guhr A, Kurtz A, et al. Human embryonic and induced pluripotent stem cell research trends: complementation and diversification of the field. Stem Cell Reports. 2015;4(5):914–925.
- Seltmann S, Lekschas F, Müller R, et al. hPSCreg–the human pluripotent stem cell registry. Nucleic Acids Res. 2016;44(D1):D757–763.
- Kobold S, Guhr A, Mah N, et al. A manually curated database on clinical studies involving cell products derived from human pluripotent stem cells. Stem Cell Reports. 2020;15(2):546–555.
- Petkova R, Zhelev N, Pankov R, et al. Individual capacity for repair of DNA damage and potential uses of stem cell lines for clinical applications: a matter of (genomic) integrity. Biotechnol Biotechnol Equip. 2018;32(6):1352–1358.
- Ben M'Barek K, Bertin S, Brazhnikova E, et al. Clinical-grade production and safe delivery of human ESC derived RPE sheets in primates and rodents. Biomaterials. 2020;230:119603.
- Rehakova D, Souralova D, Koutna I. Clinical-grade human pluripotent stem cells for cell therapy: characterization strategy. IJMS. 2020;21(7):2435.
- Tilgner K, Neganova I, Moreno-Gimeno I, et al. A human iPSC model of ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors. Cell Death Differ. 2013;20(8):1089–1100.
- Hare I, Gencheva M, Evans R, et al. In vitro expansion of bone marrow derived mesenchymal stem cells alters DNA double strand break repair of etoposide induced DNA damage. Stem Cells Int. 2016;2016:8270464.
- Gant VU, Junco JJ, Terrell M, et al. Enhancer polymorphisms at the IKZF1 susceptibility locus for acute lymphoblastic leukemia impact B-cell proliferation and differentiation in both down syndrome and non-Down syndrome genetic backgrounds. PLoS One. 2021;16(1):e0244863. DOI: 10.1371/journal.pone.0244863
- Rogers JH, Gupta R, Reyes JM, et al. Modeling IKZF1 lesions in B-ALL reveals distinct chemosensitivity patterns and potential therapeutic vulnerabilities. Blood Adv. 2021;5(19):3876–3890.
- Simara P, Tesarova L, Rehakova D, et al. DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing. Stem Cell Res Ther. 2017;8(1):73.
- di Val Cervo PR, Besusso D, Conforti P, et al. hiPSCs for predictive modelling of neurodegenerative diseases: dreaming the possible. Nat Rev Neurol. 2021;17(6):381–392.
- Al-Khalaf MH, Blake LE, Larsen BD, et al. Temporal activation of XRCC1-mediated DNA repair is essential for muscle differentiation. Cell Discov. 2016;2:15041.
- Regent F, Morizur L, Lesueur L, et al. Automation of human pluripotent stem cell differentiation toward retinal pigment epithelial cells for large-scale productions. Sci Rep. 2019;9(1):10646.
- Reynolds L. The success of stem cell transplantations and the potential post-transplantation complications may be dependent, among other factors, on the capacity of the recipient and the transplanted cells to repair DNA damage. BD. 2016;19:e9076.
- Khalil HS, Petkova R, Zhelev N. Differential genetic advantages in youth and in aging, or how to die healthy. Biotechnol Biotechnol Equip. 2012;26(1):2703–2711.
- Chelenkova P, Petkova R, Chamova T, et al. The fine art of vascular wall maintenance. Carriership of XPC, TP53 and APOE polymorphisms may be a risk factor for cerebral vascular accidents in the Bulgarian population. Biotechnol Biotechnol Equip. 2018;32(6):1558–1566.
- Petkova R, Chelenkova P, Georgieva E, et al. What’s your poison? Impact of individual repair capacity on the outcomes of genotoxic therapies in cancer. Part I—role of individual repair capacity in the constitution of risk for late-onset multifactorial disease. Biotechnol Biotechnol Equip. 2013;27(6):4208–4216.
- Arabadjiev B, Petkova R, Nonchev S, et al. Derivation of human embryonic stem cell line from discarded IVF morula. Compt Rend Bulg Acad Sci. 2010;63(12):1765–1770.
- Foley GE, Lazarus H, Farber S, et al. Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer. 1965;18(4):522–529.
- Schneider U, Schwenk H, Bornkamm G. Characterization of EBV-genome negative "null" and "T" cell lines derived from children with acute lymphoblastic leukaemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer. 1977;19(5):621–626.
- Krüger S, Bier A, Engel C, German Hereditary Non-Polyposis Colorectal Cancer Consortium, et al. The p53 codon 72 variation is associated with the age of onset of hereditary non-polyposis colorectal cancer (HNPCC). J Med Genet. 2005;42(10):769–773.
- Khan SG, Metter EJ, Tarone RE, et al. A new xeroderma pigmentosum group C poly(at) insertion/deletion polymorphism. Carcinogenesis. 2000;21(10):1821–1825.
- Chen P, Wiencke J, Aldape K, et al. Association of an ERCC1 polymorphism with adult-onset glioma. Cancer Epidemiol Biomarkers Prev. 2000;9(8):843–847.
- López-Cima MF, González-Arriaga P, García-Castro L, et al. Polymorphisms in XPC, XPD, XRCC1, and XRCC3 DNA repair genes and lung cancer risk in a population of Northern Spain. BMC Cancer. 2007;7:162.
- Schmitz C, Lindpaintner K, Verhoef P, et al. Genetic polymorphism of methylenetetrahydrofolate reductase and myocardial infarction. A case-control study. Circulation. 1996;94(8):1812–1814.
- Tan SM, Wang ST, Hentze H, et al. A UTF1-based selection system for stable homogeneously pluripotent human embryonic stem cell cultures. Nucleic Acids Res. 2007;35(18):e118.
- Seigel GM, Hackam AS, Ganguly A, et al. Human embryonic and neuronal stem cell markers in retinoblastoma. Mol Vis. 2007;13:823–832.
- Klijn C, Durinck S, Stawiski EW, et al. A comprehensive transcriptional portrait of human cancer cell lines. Nat Biotechnol. 2015;33(3):306–312.
- Laumann R, Jücker M, Tesch H. Point mutations in the conserved regions of the p53 tumour suppressor gene do not account for the transforming process in the jurkat acute lymphoblastic leukemia T-cells. Leukemia. 1992;6(3):227–228.
- Rafki N, Liautaud-Roger F, Devy L, et al. P53 protein expression in human multidrug-resistant CEM lymphoblasts. Leuk Res. 1997;21(2):147–152.
- Nelson HH, Wilkojmen M, Marsit CJ, et al. TP53 mutation, allelism and survival in non-small cell lung cancer. Carcinogenesis. 2005;26(10):1770–1773.
- Marín MS, López-Cima MF, García-Castro L, et al. Poly (at) polymorphism in intron 11 of the XPC DNA repair gene enhances the risk of lung cancer. Cancer Epidemiol Biomarkers Prev. 2004;13(11 Pt 1):1788–1793.
- Dai QS, Hua RX, Zhang R, et al. Poly (at) deletion/insertion polymorphism of the XPC gene contributes to urinary system cancer susceptibility: a meta-analysis. Gene. 2013;528(2):335–342.
- Wu H, Li S, Hu X, et al. Associations of mRNA expression of DNA repair genes and genetic polymorphisms with cancer risk: a bioinformatics analysis and Meta-analysis. J Cancer. 2019;10(16):3593–3607.
- Zhuo Z, Miao L, Hua W, et al. He J genetic variations in nucleotide excision repair pathway genes and hepatoblastoma susceptibility. Int J Cancer. 2021;149(9):1649–1658.
- Zhu Y, Yang H, Chen Q, et al. Modulation of DNA damage/DNA repair capacity by XPC polymorphisms. DNA Repair (Amst). 2008;7(2):141–148.
- Woelfelschneider A, Popanda O, Lilla C, et al. A distinct ERCC1 haplotype is associated with mRNA expression levels in prostate cancer patients. Carcinogenesis. 2008;29(9):1758–1764.
- Sturgis EM, Dahlstrom KR, Spitz MR, et al. DNA repair gene ERCC1 and ERCC2/XPD polymorphisms and risk of squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg. 2002;128(9):1084–1088.
- Yin Z, Zhou B, He Q, et al. Association between polymorphisms in DNA repair genes and survival of non-smoking female patients with lung adenocarcinoma. BMC Cancer. 2009;9(1):439.
- Skjelbred CF, Saebø M, Wallin H, et al. Polymorphisms of the XRCC1, XRCC3 and XPD genes and risk of colorectal adenoma and carcinoma, in a norwegian cohort: a case control study. BMC Cancer. 2006;6:67.
- Vodicka P, Kumar R, Stetina R, et al. Genetic polymorphisms in DNA repair genes and possible links with DNA repair rates, chromosomal aberrations and single-strand breaks in DNA. Carcinogenesis. 2004;25(5):757–763.
- Chai F, Liang Y, Chen L, et al. Association between XRCC3 Thr241Met polymorphism and risk of breast cancer: meta-analysis of 23 case-control studies. Med Sci Monit. 2015;21:3231–3240.
- Michalska MM, Samulak D, Romanowicz H, et al. Association between single nucleotide polymorphisms (SNPs) of XRCC2 and XRCC3 homologous recombination repair genes and ovarian cancer in polish women. Exp Mol Pathol. 2016;100(2):243–247.
- Avci H, Ergen A, Bireller ES, et al. A strong relationship between oral squamous cell carcinoma and DNA repair genes. Biochem Genet. 2017;55(5–6):378–386.
- Wilcken B, Bamforth F, Li Z, et al. Geographical and ethnic variation of the 677C > T allele of 5,10 ethylenetetrahydrofolate reductase (MTHFR): findings from over 7000 newborns from 16 areas worldwide. J Med Genet. 2003;40(8):619–625.
- Petrone I, Bernardo PS, Dos Santos EC, et al. MTHFR C677T and A1298C polymorphisms in breast cancer, gliomas and gastric cancer: a review. Genes (Basel). 2021;12(4):587.
- Arancibia T, Morales-Pison S, Maldonado E, et al. Association between single-nucleotide polymorphisms in miRNA and breast cancer risk: an updated review. Biol Res. 2021;54(1):26.
- Chicheva Z, Chelenkova P, Petkova R, et al. Children of the sun, children of the moon—a mini-panel for assessment of inter-individual variation between the capacity of healthy individuals to repair everyday genotoxic insults. Biotechnol Biotechnol Equip. 2012;26(4):3142–3147.
- Chelenkova P, Petkova R, Chamova T, et al. Homozygous carriership of the wildtype allele of the XPCins83 polymorphism is an independent protective factor against cerebrovascular incidents in the bulgarian population. Compt Rend Acad Bulg Sci. 2014;67(2):263–268.
- Abbasalizadeh S, Baharvand H. Technological progress and challenges towards cGMP manufacturing of human pluripotent stem cells based therapeutic products for allogeneic and autologous cell therapies. Biotechnol Adv. 2013;31(8):1600–1623.
- Sverdlov ED, Mineev K. Mutation rate in stem cells: an underestimated barrier on the way to therapy. Trends Mol Med. 2013;19(5):273–280.