The effect of reprogramming method, source cell type and long-term cell culture on genomic stability of human induced pluripotent stem cells
| Autoři | |
|---|---|
| Rok publikování | 2015 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | Human induced pluripotent stem cells (hiPSCs) possess a great potential for clinical application. However, previous studies revealed the genomic instability of these cells. The reprogramming process itself may contribute to the mutational load in hiPSCs and subsequent in vitro culturing is also related to genomic aberrations increase. Various methods for iPSC generation were established and the main effort has been focused on reprogramming efficiency. In our study we try to figure out how reprogramming method, source cell type and long-term cell culture influences the genomic stability of hiPSCs. In our laboratory we established hiPSC clones from different source cells (fibroblasts or CD34+ blood progenitors) by three reprogramming methods: STEMCCA lentivirus, Sendai virus or episomal vectors. The pluripotency of our hiPSCs was verified by differentiation into all three germ layers and by teratoma assay. In order to study genomic integrity, we monitored DNA damage response (DDR). Phosphorylated form of histone H2AX (g-H2AX) and protein 53BP1 play key role in DDR mechanism and mark DNA lesions throughout the genome. The levels of g-H2AX and 53BP1 were determined in source cells, hiPSCs in low passage and hiPSCs in high passage. The immunofluorescence analysis revealed the differences in spontaneously occurring foci numbers among our hiPSC lines and variations were also found between low and high passages. Moreover, the samples differ in their capacity to response to ionizing radiation. Expectedly, the two proteins were extensively co-localized. We hypothesize that observed variations in DDR will correlate with the genomic aberrations, including duplications and deletions. Therefore, CGH microarray technology will be employed to detect copy number variations that may result from impaired DDR. The genomic stability is one of the major safety concerns of hiPSCs and must be addressed before transfer of this technology into clinical therapy. |
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