Altered genome induced immune response of iPSCs

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 suggested that the lower immunogenicity of UMCs could persist after cell reprogramming and further differentiation. This discovery goes in the line with the AGIIR hypothesis: that generation of functional lineages with lower immunogenicity from iPSCs strongly depends on genomic and epigenetic stability.It is unclear whether iPSC-derived cells can be immunogenic at different extents as a consequence of aberrations acquired, and if the genetic alterations may affect or not the transplantation potential. Thus, it appears to be of great relevance to estimate the immunogenicity of clinical valuable cells, as well as the tissue specific propensity to become immunogenic depending on the number and type of cumulated defects. Work from our group demonstrated abnormal toll like receptor 3 (TLR3) gene methylation and expression in iPSCderived cells, suggesting dysregulated innate immune responses (Requena et al., 2019).Different immunogenic predispositions of iPSCs could depend on the cell type they are differentiated to (Figure 3). This idea was first proposed by Dr.'s Xu group (Zhao et al., 2011), and was later reviewed (Cao et al., 2014).In the race to the clinic, the potential immunogenicity of iPSC-derived cells has been largely overlooked. A number of publications have described mechanisms inferred from genetic and epigenetic instability to predict potentially elicit immune responses, and others have demonstrated established mechanisms observed in iPSC-derived cells, including our own work in 2019 on epigenetic changes in the toll-like 3 receptor (TLR3) (Requena et al., 2019) (Table 1). Aberrant or ectopic expression of selfantigens.Genetic or epigenetic dysregulation during reprogramming causes normally silent selfantigens in target cells to be expressed in iPSC-derived cells, may lead to presentation of self-peptides that might break tolerance and trigger T cell responses.Hypothetical Zhao et al., 2011;Fairchild, 2010;Cao et al. 2014;Araki et al., 2013 Overexpression of lineage-specific antigens.Copy number changes or selective clonal expansion during culture lead to overexpression of tissue-specific proteins, potentially triggering immune recognition. Reprogramming and differentiation affect molecules involved in antigen display (e.g., MHC-I and β2-microglobulin expression), altering the peptide repertoire presented to T cells.Established Pick et al., 2012;Boyd et al., 2009;Drukker et al., 2002 Abnormalities in innate immune sensing.Epigenetic changes affect innate immune pathways (e.g., TLR3) influencing cytokine signaling and immune activation.Established Requena et al., 2019 As discussed above, the reprogramming process itself has a major impact on the genetic landscape that could impact autologous cell therapy approaches, suggesting that a potential immunogenic role of autologous cells transplantations could have been underestimated. More than a decade ago, it was proposed that genetic and epigenetic aberrations acquired during reprogramming could increase cell immunogenic potential (Fairchild et al., 2010).The altered genome induced immune response (AGIIR) hypothesis postulates that cell reprogramming-derived genetic and epigenetic alterations may lead to immune dysregulation in certain cell types (Figure 2). Some genomic alterations may affect genes or promoters involved in the differentiation of iPSCs to neurons but not to hepatocytes, provoking differentiation to immunogenic neurons but normal hepatocytes. Whether the most frequent alterations in pluripotent cells, like duplications on chromosomes 1, 12, 17 and X or the specific amplification in 20q, are destined to be immunogenic is yet to be determined. It seems likely that most iPSCs genomic aberrations are going to be harmless and only a few abnormalities will actually be hazardous. However, it is still an open question to know which kind of tissues differentiated from iPSCs can be immunogenic due to cell type-specific aberrations, such as cells abnormally expressingHormad1 and Zg16 genes (Zhao et al., 2011) or cells carrying other specific genetic and/or epigenetic aberrations still unknown. Zhao et al. in 2011 demonstrated that after injecting retrovirally reprogrammed iPSCs in syngeneic recipients, induced T-cell-dependent immune response prevents the formation of teratomas in mice (Zhao et al., 2011). Teratomas that did not regress were infiltrated with CD4 + T cells with apparent necrosis within parts of the tissue.This rejection was not observed after injection of syngeneic mouse ESCs (mESCs).Regarding rejection of transplanted allogeneic cells and organs, the main immune response et al., 2010;Robertson et al., 2007;Drukker et al., 2002).While human iPSC-derived cells immune response may appear minimal, immunogenicity may play a major role in their potential applicability in the clinic, as some subsets of genetic and epigenetic aberrations might have the potential to generate immune responses. The proposed Altered Genome Induced Immune Response (AGIIR) hypothesis might explain some of the immunogenic responses reported with iPSC-derived cells in the literature, such as T cell infiltration in teratomas (Zhao et al., 2011) and immunogenic cardiomyocytes (Araki et al., 2013).Finally, before clinical application of iPSCs derivatives becomes a reality, it is crucial to study the influence of these alterations accurately and assess the risk stratification for immunogenicity. Classifying the major genetic and epigenetic alterations that may elicit an immune response should be included as part of a standard operating procedures (SOPs) for the clinical use of human iPSCs.