Altered genome induced immune response of iPSCs

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The most frequent chromosome duplications (whole chromosome and subchromosomal regions) in pluripotent cells are in autosome chromosomes 1, 12, 17 and sex chromosome X.Amplifications in 20q region have been detected in 34% of ESC and iPSC lines examined (Amps et al., 2011;Taapken et al., 2011;Mayshar et al., 2010). Trisomy of chromosome 12 is the most recurrent abnormality in both ESC (42.6%) and iPSCs (32.9%) (Mayshar et al., 2010;Taapken et al., 2011). Interestingly, many chromosomal abnormalities found in ESC are also found in iPSCs. However, while chromosome 8 gains are more likely to be found in iPSCs, chromosome 17 gains are more likely to be found in ESC (Taapken et al., 2011;Ben-David et al., 2011;Martins-Taylor et al., 2011). High resolution single nucleotide polymorphism (SNP) analysis mapping of ESCs and iPSCs found common subchromosomal duplications in chromosome 20q, in genes conferring cell growth or survival advantage, such as BCL2L1 (20Q11.21). BCL2L1 enhances ESC survival giving therefore a selective advantage by attenuation of apoptosis; or mir1825, which has over 400 predicted targets, triggering suppression of apoptosis and cell growth enhancement (Bai et al., 2012). Importantly, recent work has demonstrated that iPSC culture introduces mutations in the BCOR gene that can affect the differentiation process, particularly to neurons and may impact other cell functions that could include the immune system, currently under investigation (Rouhani FJ et al, 2022;Puigdevall P, et. Al., 2023).Gene copy number variation (CNV) by itself is not necessarily a high-risk trait. A mounting number of studies have demonstrated that somatic mosaicism of ordinary cells is a normal characteristic of the human body (Chen et al., 2013;Lupski et al., 2013;Poduri et al., 2013;Biesecher et al., 2013). However, human iPSCs have a higher number of subchromosomal CNV than ESC (Laurent et al., 2011;Martins-Taylor et al., 2011;Hussein et al., 2011). Earlypassage iPSCs are characterized by a huge incidence of CNV compared with parental fibroblasts. These alterations, especially copy number losses, are usually negatively selected in culture. Recently it has been described that CNV can be profiled by using a high-density DNA methylation array with the same sensitivity of SNP platforms (Feber et al., 2014). The most recurrent CNV hotspot is amplification of the gene-rich locus at the long arm 20q11.21. It is estimated to be present in approximately 14.5% of ESC and iPSC lines (Matins-Taylor et al., specific culture conditions to maintain homogeneous genomically stable populations and a safe passage number threshold cannot be determined.Genomic alterations can also be selected for during differentiation of ESCs and iPSCs. For example, an abnormal subpopulation of ESCs with multiple duplications in chromosome 20, after only 5 days, was selected in a cardiac differentiation experiment to cardiomyocytes (Laurent et al., 2011). Interestingly, multipotent adult stem cells also show frequent typical chromosomal abnormalities, like duplication of chromosome 19 in neural stem cells (NSCs) or a deletion of chromosome 13 in mesenchymal stem cells (MSCs) (Ben-David et al., 2011).Regarding point mutations, exome sequencing has shown that 74% of mutations detected in iPSCs are generated during reprogramming, 19% pre-existed in parental fibroblasts, and only 7% are caused by in vitro maintenance (Ji et al., 2012). Nevertheless, selection of pre-existing subpopulations of mutant parental fibroblasts during reprogramming was found to explain this high percentage (Young et al., 2012).Reprogramming to human iPSCs can induce epigenetic anomalies (Allegrucci C, 2011;Ferguson-Smith, 2011;Meissner, 2010;Planello et al., 2014). Epigenetic alterations refer to alterations in patterns of (a) gene imprinting, (b) DNA methylations and (c) histone modification.Imprinting is the epigenetic silencing found in some alleles of specific genes depending on a parent-of-origin specific manner. Typically, alterations in imprinting provide growth advantages for pluripotent cells maintained in culture because many imprinted genes are known to regulate growth during embryonic development (Piedrahita et al., 2011). A large-scale comparison of ESC, iPSCs, somatic tissues and primary cell lines demonstrated that pluripotent cells are characterized by a high level of variation in the methylation status of a subset of imprinted genes (Nazor et al., 2012). Genetic variation and instability were discovered in the imprinting status of a subset of genes in pluripotent cell lines, such as the paternally imprinted genes H19 and the maternally expressed 3 (MEG3) tumour suppressor (International Stem Cell Initiative, 2007).DNA methylation in pluripotent cell lines is typical for a subset of imprinted and developmental genes, for instance the alteration in methylation of the tumour suppressor RAS association domain family member 1 (RASSF1) (Papaspyropoulos et al., 2018), suggesting a positive selection pressure to culture induced methylation changes. Human iPSCs have been reportedto have increased levels of DNA methylations, which are aberrant and different from ESC during early passages. However, during prolonged culturing, the level of DNA methylation gradually becomes even (Nishino et al., 2011). Moreover, studies with iPSC-derived neurons suggest that many DNA methylation differences between iPSCs and ESCs are largely normalized upon differentiation (de Boni et al., 2018). Furthermore, it has been shown that abnormal methylation patterns in iPSCs are influenced by the choice of reprogramming factors, with different factor combinations leading to distinct patterns of methylation error (failure to demethylate vs. failure to methylate) (Planello et al., 2014).Human iPSCs have increased levels of H3K27me3 and several studies have demonstrated differences with histone 3 trimethylations marks between ESC and iPSCs (Doi et al., 2009;Guenther et al., 2010;Deng et al., 2009). Other studies demonstrated that lysine 9 (H3K9me3) rather than lysine 27 (H3K27me3) is highly modified (Hawkins et al., 2009). Lysine 4 (H3K4me3) variation patterns were found to be similar (Guenther et al., 2010). In addition, such changes were also reflected at the transcript level with changes in the expression of multiple genes involved in developmental and epigenetic processes.The immunogenicity of iPSC-derived cells is a subject of ongoing research. Guha et al. found that transplanted cells derived from syngeneic iPSCs were not rejected after transplantation (Ghua et al., 2013). Also, Araki R. et al., compared the immunogenicity of skin and bone marrow cells derived from mouse iPSCs to the immunogenicity of ESC-derived tissue and did not observe any differences between the two groups, finding limited immunogenicity in both cases (Araki et al., 2013). This support the idea that autologous iPSCs could be applied for cell replacement therapies without eliciting immune rejection. However, and revealingly, in the same study it was shown that cardiomyocytes derived from these same iPSCs elicited an immunogenic response, as observed by increased T-cell infiltration (Araki et al., 2013). On the other hand, Morizane et. al., found that autologous transplantation of iPSC-derived cells generated a minimal immune response compared with allografts in non-human primate brains in the absence of immunosuppression (Morizane et al., 2013). They suggested that immunosupression was not necessary for autologous transplantation of iPSC-derived neural cells in the brain. In contrast, Liu et. al., differentiated iPSCs derived from human umbilical cord mesenchymal stem cells (UMCs) or skin fibroblasts (SFs) into neural progenitor cells (NPCs) and analysed their immunogenicity. They reported a lower immunogenicity of NPCs differentiated from iPSCs derived from UMCs than from SFs (Liu et al., 2013), retaining a low immunogenicity as the parental UMCs. Hence, the authors

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