Global Proteomic and Methylome Analysis in Human Induced Pluripotent Stem Cells Reveals Overexpression of a Human TLR3 Affecting Proper Innate Immune Response Signaling

Abstract

When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)‐derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC‐derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC‐derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC‐NSC functions to suppress NF‐KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC‐derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC‐derived cells calling for screening of human iPSC‐derived cells for TLR3 expression levels before applications. Stem Cells 2019;37:476–488

iPS-Cell Technology and the Problem of Genetic Instability—Can It Ever Be Safe for Clinical Use?

“…Issues such as the relationship between alterations in the sequences (e.g., de novo mutations) at certain regions of the genome and inflammation or immune response has not been considered due to the relatively short follow-up time post transplantation and the low number of clinical studies to date. How mutations, epigenetic changes or alterations in non-coding regions are related to inflammation remains poorly understood [87]. Nevertheless, potential tests for immunogenicity of autologous hiPSC-derivates are already beginning to emerge in the form of screening for markers associated with aberrant immune signaling (e.g., overexpression of TLR3 short-isoform [87]) or directly for immune response (e.g., use of humanized mouse models [88]).…”

“…How mutations, epigenetic changes or alterations in non-coding regions are related to inflammation remains poorly understood [87]. Nevertheless, potential tests for immunogenicity of autologous hiPSC-derivates are already beginning to emerge in the form of screening for markers associated with aberrant immune signaling (e.g., overexpression of TLR3 short-isoform [87]) or directly for immune response (e.g., use of humanized mouse models [88]).…”

“…Other work suggests that the T-cell response to undifferentiated iPSC is different from that mounted against their mature derivatives [86]. Furthermore, proteomic and epigenetic study suggests atypical innate immune response signaling in iPSC-derived cells [87]. In contrast, there are indications that autologous-derived cells are immunogenic, although this varies with tissue type (but not injection site); for example, hiPSC-derived-smooth muscle cells were highly immunogenic, whereas hiPSC-derived RPE cells were not [88].…”

The use of induced pluripotent stem cells in domestic animals: a narrative review

“…The investigation of -omics in iPSCs can assist in confirming how completely iPSCs have been reprogrammed to an undifferentiated stem cell state and resemble ESCs. Studying proteins and sites of methylation have clinical applications in autologous cell replacement therapy [ 169 ]. Aside from one study investigating the effects of epigenetic modifiers on silencing on exogenous transcription in piPSCs [ 65 ], epigenetics is an unexplored area of iPSC research in domestic species.…”

Episomal Induced Pluripotent Stem Cells: Functional and Potential Therapeutic Applications

“…The human cells reported to have been successfully reprogrammed into EiPSCs include fibroblasts, epithelial cells, keratinocytes, mononuclear cells from adult peripheral blood, cord blood cells, amniotic fluid stem cells, mesenchymal stromal cells, lymphoblasts, lamina propria progenitor cells from oral mucosa, and urothelial cells obtained from urine. A summary of these reported human EiPSC sources is shown in Table 1 8,20,21,32 –43,47 –52,55,56,58,60 …”