Embryonic stem cells (ESCs) are considered pluripotent and are capable to give rise to all types of somatic cells. This ability of ESCs can be utilized in treating certain diseases. ESC like pluripotent stem cells were created successfully in a research work published in 2006 and they were termed as induced pluripotent stem cells or iPSCs. The iPSCs were possible when four transcription factors called Oct4, c-Myc, Sox2 and Klf4 were introduced into somatic cells of mice. The newly created iPSCs were same as ESCs in their gene expression, morphology, In Vitro differentiation capacity and in epigenetic status. Many studies say that iPSCs are not beneficial clinically due to their genomic instability.
The research work done recently reveals that iPSCs consist of chromosomal anomalies like duplications in chromosome 12 of human iPSCs. The reprogramming of iPSCs is always considered incomplete. The epigenetic and transcriptional properties of these cells are affected by donor cells. The lack of p53 was found to be useful for enhancing the chances of DNA damage, reprogramming, and genomic instability. Therefore, there are higher chances for genomic abnormalities to be present in the iPSCs. Hence, it is necessary to concentrate more on the iPSC genomic status prior to their usage in clinical treatments.
To study the specific reason for genomic instability, DNA damage was introduced into iPSCs through ionizing radiation (IR) and the difference in the ability of iPSCs in DNA damage repair compared to that of mouse embryonic fibroblast cells (MEF) was observed. This paper discusses various tests and test results associated with iPSCs DNA damage repair capacity from various studies.
Results of the studies
Low DNA damage repair ability of irradiated iPSCs
To test the hypothesis that genomic instability of iPSCs was due to their low DNA damage repair capability, the iPSC cell line 3FB4-1 was selected. The cell line 3FB4-1 was derived from somatic MEF cells of mice and they were irradiated to generate double strand DNA breaks. The dsDNA breaks can result in apoptosis, genome abnormalities and senescence.
Through immunoblotting analysis, it is found that ataxia-telangiectasia-mutated or ATM which is vital in triggering the DNA repair was autophosphorylated and activated due to irradiation. The histone variant gamma-H2AX is a DSB marker which alters its levels in 3FB4-1 and MEF cells. The slight increase in gamma-H2AX focus number of 3FB4-1 cell line than that of MEF cells gives an evidence for low DNA damage repair capacity of iPSC cells.
Whole genome sequencing
Single nucleotide variation analysis and genome DNA sequencing was performed for MEF cells and 3FB4-1 cells, both prior to irradiation and after irradiation. Gelatin resuspension method was used for collecting 3FB4-1 cells. The DNA length in each of the four samples was greater than 64 Gb. Mutations in each of the samples were identified.
iPSCs recorded more point mutations
The single nucleotide variation (SNV) analysis could reveal the presence of point mutations in iPSCs. Samtools Sequence Map Tool and Burrows-Wheeler Alignment Tool were utilized for scanning the mouse reference genome. Every sample was detected with nearly 2.9 to 4.2 X 106 SNVs. The variation in the sequences of 3FB4-1 (IR+) and 3FB4-1 (IR-) cells were directly compared with that of MEF (IR+) and MEF (IR-) cells. Nearly 9,08,296 SNVs were present in 3FB4-1 cells after ionized radiation and 1,83,518 SNVs were present in MEF cells.
The mutation number in 3FB4-1 cells increased considerably after irradiation from 4,94,616 to 9,36,728. The mutation number in MEF cells increased from 1,243,249 to 1,312,621. When observed individually, 3FB4-1 cells had 3,02,226 single nucleotide variations that were common for IR+ and IR- cells. But, 6,34,502 SNVs were identified from 3FB4-1 cells with IR. In MEF cells (IR+), 3,27,625 SNVs were recovered while 9,84,996 SNVs were recovered commonly from IR+ and IR- MEF cells. From this data, it can be understood that more number of SNVs were observed in irradiated 3FB4-1 cells than in irradiated MEF cells.
The increase in the number of point mutations was noticeable in each chromosome of irradiated 3FB4-1 cells while nearly 3 to 6 percent increase was observed in irradiated MEF cells.
Point mutations in known regions of the genome
The functional importance of these point mutations was investigated in both of the irradiated cells. The results of the experiments revealed that many of these point mutations were present in the introns as well as in intergenic regions. Some of them were present in the untranslated regions of 5' and 3' ends as well as in the sites of splicing. After irradiation, MEF cells and 3FB4-1 cells were observed to possess 949 and 2256 identical point mutations respectively. Number of non- identical point mutations that were observed in irradiated 3FB4-1 and MEF cells were 1454 and 724 respectively.
The noteworthy changes observed in 3FB4-1 (IR+) cells comply with the findings of several studies that iPSCs have lower DNA damage repair capacity and these cells are observed to be more sensitive to any damage to the DNA.
Minjie Zhang, Caiyun Yang, Huixian Liu, Yingli Sun. Induced pluripotent stem cells are sensitive to DNA damage. Genomics Proteomics Bioinformatics 11 (2013), 320-326.
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