Promoter methylation in iPS cells
The transplantation of neurons derived from induced pluripotent stem cells (iPSCs) presents therapeutic potential for the treatment of neurological diseases, but functional integration of transplanted iPSC-derived neurons into host neural networks remains controversial. Optogenetic and chemogenetic tools offer means to assess such integration, but constructing modifiable iPSC-derived neurons requires efficient gene editing. Here, we used CRISPR/Cas9 (targeting AAVS1 safe harbor) and PiggyBac transposon systems to insert optogenetic and chemogenetic receptor (ChR2/hM4Di) into human iPSC cells. While both systems successfully integrated genes into the genomes of HEK 293T and iPSC cells, expression of the receptors was detected only in HEK293T cells. Bisulfite sequencing revealed extensive methylation of the TRE3G BI promoter (95.3–98.2%) in iPSC cells, contrasting low methylation (5.9%) in HEK 293T. For PiggyBac, methylation of CMV/EF1a promoters in iPSC exhibits integration site-dependent variability (0–95.2%). Notably, even hypomethylated clones failed to show gene expression, suggesting the involvement of non-epigenetic regulatory mechanisms. Differentiation into neural stem cells did not reverse methylation or restore protein expression. Our findings demonstrate that CRISPR/Cas9 and PiggyBac systems enable the integration of optochemical receptor genes into iPSCs. However, epigenetic and non-epigenetic gene silencing mechanisms pose barriers to direct transgene modification in iPSCs.