Genomic instability of human pluripotent stem cells (iPSCs) was first documented in 2004, reporting karyotypic abnormalities in human embryonic stem cells (ESCs), including trisomy of chromosome 12 (1, 2). Several reviews have been written focusing on ESCs and iPSCs (3) genomic and epigenomic instability (4–8). It is widely accepted that cell reprogramming to iPSCs can induce both genetic and epigenetic defects in iPSCs (Figure 1) (9–12). Analysis of non-integrative virus-free methods, such as mRNA transfection (13) and episomal vectors (14) found that the reprogramming method impacts on genomic changes in iPSCs, with mRNA reprogramming method generating less genomic instability (14, 15). Genetic alterations range from single nucleotide point mutations to whole chromosome aneuploidies (including mosaic) or sub chromosomal aberrations, including gene duplications and deletions (14, 16–18).

Genomic changes can occur during the cell reprogramming process to pluripotency, long term culture of iPSCs or during differentiation of iPSCs to various cell types, but not limited, such as neurons, cardiomyocytes and beta islets cells (19). Interestingly, late passage iPSCs were twice as likely to encumber genomic changes compared with early passage cells (33% compared with 14%), as reported in 2011 in a large-scale study of more than hundred iPSCs lines (16). This result points to the key fact that selective pressure plays an important role in favoring accumulation of genomic alterations that confers growth advantage.

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 (56). 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 (57). 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 (57). 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 (58). 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 analyzed their immunogenicity. They reported a lower immunogenicity of NPCs differentiated from iPSCs derived from UMCs than from SFs (59), 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 iPSC-derived cells, suggesting dysregulated innate immune responses (60).

Different immunogenic predispositions of iPSCs could depend on the cell type they are differentiated to (Figure 2). This idea was first proposed by Dr.’s Xu group (61), and was later reviewed (62).

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) (60) (Table 1).

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 (63).

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 expressing Hormad1 and Zg16 genes (61) or cells carrying other specific genetic and/or epigenetic aberrations still unknown. (61) demonstrated that after injecting retrovirally reprogrammed iPSCs in syngeneic recipients, induced T-cell-dependent immune response prevents the formation of teratomas in mice (61). 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 involves the major histocompatibility complex-I (MHC-I), expressed on every nucleated cell in the body, whose function is to present foreign antigens to T cells. Pick et al., demonstrated that during reprogramming, iPSCs downregulated expression of human leukocyte antigen (HLA)-A/B/C and β2 microglobulin (β2M) (64), the two components of MHC class I (MHC-I). Their results showed very low expression levels of MHC-I proteins on the surface of ESCs. During differentiation of ESCs, high levels of MHC-I expression are observed, resulting in an increase in immunogenicity in transplanted ESC-derived cells (65–67).

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 (61) and immunogenic cardiomyocytes (57).

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.