Genetic instability of human pluripotent cells
A.S. Grigoryan, "Cell transplantology and tissue engineering"
based on: Dynamic changes in the copy number of pluripotency and cell proliferation genes
in human ESCs and iPSCs during reprogramminh and time in culture.
(Laurent L.C. et al., Cell Stem Cell 2011; 8: 106-18)
The ability to self–renew and differentiate into any cell types of an adult organism makes human pluripotent cells (embryonic stem cells – ESCs and induced pluripotent stem cells - iPSCs) a potential source of material for cell therapy. Today, cell therapy is the subject of numerous studies designed to assess its safety. Most scientists have been focusing their attention on the possible tumorigenicity of the cells used for many years [1].
Since genetic disorders can initiate malignant degeneration, it is extremely important that cell cultures intended for use in the clinic are genetically stable. Some human ESC lines, as has been shown in several studies, are prone to acquiring aneuploidy during cultivation [2-6], and the most common chromosomal changes in ESC, such as trisomies on chromosomes 12 and 17, are also characteristic of malignant embryonic tumor cells [7-9]. Aneuploidies can be detected by karyotyping, but more difficult to detect subchromosomal rearrangements involving groups of several nucleotides or even single nucleotide substitutions can also be the cause of cell malignancy. Such disorders can be detected only by comparative genomic hybridization (CGH analysis) or analysis of single nucleotide substitutions (single-nucleotide polymorphism, SNP analysis) [10-12].
In January 2011, the journal Cell Stem Cell published the results of the first large-scale study devoted to detailed SNP analysis of a large number of ESC lines (69 lines, 130 samples), iPSCs (37 lines, 56 samples), somatic cells (11 lines, 11 samples), primary cultures (41 lines, 41 samples) and 20 different human tissues (67 samples). In parallel, 2 SNP analysis algorithms were used: CNVPartition (Illumina, Inc., USA) and Nexus (Biodiscovery, Inc., USA) to exclude possible errors and obtain the most objective data. All results were verified by quantitative polymerase chain reaction. It was found that in the DNA of ESCs and iPSCs, unlike somatic cell lines and primary cultures, there is a large number of genetic disorders. Moreover, the authors of the work identified specific regions of the genome in which disorders occur in various lines of pluripotent cells.
In ESCs, compared with differentiated cells, significantly more frequent occurrence of duplications in 152 regions of the genome was found. The most important duplications were on chromosome 12, affecting the region of the Nanog and Oct4/Pou5f1 pluripotency genes and the NanogP1 pseudogene (in 9 ESC lines), and on chromosome 20, affecting the region above the DNMT3B methyltransferase gene (in 7 ESC lines, as well as in 1 iPSC line). At the same time, part of the duplications captured the DNMT3B gene itself (in 6 ESC lines). Such duplications are characteristic of the cells of many malignant tumors [13-16]. In the process of long-term cultivation (more than 80 passages) these chromosomal changes persisted and accumulated, that is, the cells carrying them in their DNA had a selective advantage.
For iPSCs, researchers assessed the accumulation of chromosomal aberrations at three stages: immediately after their receipt (reprogramming of HDF51 embryonic fibroblasts previously subjected to thorough SNP analysis), during long-term cultivation, and after induction of the spontaneous differentiation process. It was found that iPSCs immediately after reprogramming and in the early passages (5-8 passages), unlike ESCs, are characterized not by duplications of pluripotency genes, but by deletions in regions of the genome containing tumor suppressor genes (for example, the FRS2 gene). No such disorders were found in the original fibroblast line. During long-term cultivation (25-34 passages), duplications of genes whose increased expression is associated with malignant cell degeneration also appeared in iPSCs: RHOC, NRAS, AKT3, MDM2, CTAGE4 [17]. It was also shown that in the process of spontaneous differentiation in iPSCs, there is a sharp increase in the number of genetic aberrations, and the number of cells with deletions in the regions of tumor suppressor genes and oncogenes duplications increased in culture (the researchers evaluated the cells on the 2nd and 7th days after the start of the spontaneous differentiation process). A total of 12 iPSC clones obtained from the same line of embryonic fibroblasts were evaluated, and the same pattern was observed in all cases. Thus, not only cultivation, but also differentiation turned out to be a highly selective process, during which cells with an aberrant genome gained an advantage.
The results of this in-depth and thorough research are very disturbing. Human pluripotent cells tend to accumulate genetic disorders that are not detectable by routine procedures such as karyotyping and other methods based on the use of light microscopy. It is important that the researchers have not been able to establish at what point subchromosomal abnormalities appear in the cells – both in ESC and iPSC, since the cells contained them already at the earliest passages. At the same time, the disorders were not accidental, but were repeated in various lines of ESC and iPSC, and it is safe to say that pluripotent cells are prone to accumulation of duplications in the regions of the genome containing pluripotency genes and oncogenes, as well as deletions in the regions of tumor suppressor genes. All this can cause the development of a tumor process when such cells and their derivatives enter the recipient's body, and therefore research is required aimed not only at developing methods for controlling the genetic stability of pluripotent cells, but also at creating a way to prevent genetic aberrations. It may be necessary to revise the standard methods of obtaining and conditions for cell culture, since without a guarantee of genetic stability, pluripotent cells cannot be considered as a source of differentiated cells for transplantation in the clinic.
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