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  • br Results br Discussion Controlling the chromosome

    2018-10-22


    Results
    Discussion Controlling the chromosome stability of hESCs and their derivatives remains a fundamental challenge in trials of hESC derivative-based cell therapy. In 2004, Andrew and co-workers reported that chromosomal instability during extensive hESC expansion was due to the monensin status of hESCs during in vitro culture (Draper et al., 2004). In particular, chromosomal aberrations have been frequently reported for chromosomes 12 and 17, and this phenomenon was thought to lead to tumor progression (Yang et al., 2008a). In this present study, we compared and examined abnormal cell division with multipolar spindles during aneuploid hESC cell division in in vitro culture. We confirmed that hESCs carrying a trisomy of chromosome 12 underwent aberrant cell division with multiple spindles (Fig. 1I) and provided clear evidence that cells with an aberrant chromosome 12 propagated similarly to cancer-like cells (Sluder et al., 1997; Chen and Horwitz, 2002; Brinkley and Goepfert, 1998). The common occurrence of changes in chromosome 12 during in vitro culture conditions indicates that if the condition is not monitored closely, hESCs can easily become cancer-like or embryonic carcinoma cells (Draper et al., 2004; Gertow et al., 2007). Thus, it is critical to examine the cause of the chromosome instability and develop optimized culture techniques. Figure 2A (hEB formation) shows that PECAM-expressing cells within hEBs derived from abnormal hESCs were similar to our previous work which has demonstrated that PECAM-positive cells are localized in the center of normal hEBs (Cho et al., 2007; Kim et al., 2007b). Chromosome instability with trisomy of chromosome 12 affects the proliferative potential of undifferentiated hESCs in culture but does not influence them during endothelial cell differentiation in the present study. Finally, we assessed the survival of karyotypically normal and abnormal hESC derivatives in vivo and their potential to undergo chromosomal changes after transplantation. Numerous researchers have transplanted hESC derivatives in animal models and confirmed their survival and functionality in vivo (Cho et al., 2007; Laflamme et al., 2007; Lu et al., 2007), but the potential chromosomal changes of the donor cells in vivo have not been studied. In addition, many researchers have attempted to analyze in vivo cell survival using cell tracking (Brazelton and Blau, 2005), molecular imaging (van der Bogt et al., 2006), and gene transfection (Li et al., 2008) but have failed to provide accurate results (Swenson et al., 2007; Pawelczyk et al., 2008). To overcome the above-noted problems, we established RFP-hESCs (Fig. 3) using a lentivirus system and produced a new animal model with a wound using a PE chimney for observation of transplanted cells (Fig. 4). Using our developed system together with techniques such as RFP transfection, euploid and aneuploid hESCs carrying a trisomy of chromosome 12 were induced to differentiate and analyzed for 6weeks after transplantation into an animal model. Transplanted euploid hESC derivatives maintained normal human chromosome numbers, but aneuploid hESC derivatives continued to undergo further chromosomal changes in two among the seven mice tested. These results demonstrate the importance of maintaining normal human chromosomes prior to transplantation; the safety of hESC derivatives carrying normal human chromosomes is improved compared with those carrying abnormal chromosomes. In summary, we first discovered that the chromosome instability with trisomy of chromosome 12 in hESCs caused them to divide with multiple spindles and increased the proliferative potential of undifferentiated hESCs but did not affect the differentiation of endothelial cells. Second, we found that the chromosome instability of hESC derivatives gave rise to a tumor-like tissue in vivo in the absence of teratomas, using RFP-hESCs and a newly developed wound model to analyze the fate of injected hESCs derivatives. Thus, our approach used to assess the importance of chromosome stability in hESCs revealed that the chromosome instability of hESCs with trisomy of chromosome 12 during maintenance changed the proliferative potential of hESCs via cell division with multiple spindles in vitro and their derivatives, which formed a tumor-like tissue in vivo. Based on these results, we suggest that optimized culture techniques that sustain the chromosome stability of hESCs should be required for clinical trials using hESC derivatives.