# Effective and stable gene transduction in rhesus macaque iPSCs capable of T-lineage differentiation utilizing the piggyBac system

## TL;DR

Researchers developed a method using the piggyBac system to effectively and stably transfer genes into rhesus macaque iPSCs, which can differentiate into T cells.

## Contribution

The study introduces an effective and stable gene transduction method for rhesus macaque iPSCs using the piggyBac system.

## Key findings

- The piggyBac transposon vector enables long-term gene expression in Rh-iPSCs.
- Gene-transduced Rh-iPSCs form teratomas and maintain transgene expression after differentiation into T cells.
- Differentiation efficiency of gene-transduced Rh-iPSCs is comparable to parental cells.

## Abstract

Genetically modified human induced pluripotent stem cell (iPSC)-based regenerative medicine has substantial potential in the treatment of refractory human diseases. Thus, preclinical studies on the safety and efficacy of these products are essential. Non-human primate (NHP) models such as the rhesus macaque are highly similar to humans in terms of size, lifespan, and immune system, rendering them superior models. However, effective gene transduction in rhesus macaque iPSCs (Rh-iPSCs) remains challenging. In this study, we investigated the effective gene transduction into Rh-iPSCs and its effect on differentiation efficiency.

We established a gene transduction method using the piggyBac transposon vector system. Gene transduced Rh-iPSCs were analyzed for undifferentiated markers. We did teratoma assay to check pluripotency. Gene transduced Rh-iPSCs were differentiated into hematopoietic stem and progenitor cells (HSPCs) and T-cell lineage cells. Additionally, gene transduced Rh-iPSCs were compared the differentiation efficiency with parental Rh-iPSCs.

We could establish a gene transduction method using the piggyBac transposon vector system, demonstrating high efficiency and stable transgene expression in Rh-iPSCs. These Rh-iPSCs maintained long-term gene expression while expressing undifferentiated markers. Teratoma assay indicated that these Rh-iPSCs had pluripotency. These Rh-iPSCs could differentiate into HPSCs and T cells that express transgenes. These Rh-iPSCs can differentiate into hematopoietic stem cells and T cells that express transgenes. No significant differences in efficiency of differentiation were observed between parental Rh-iPSCs and these Rh-iPSCs.

These results indicate that the piggyBac transposon vector is an excellent gene transfer tool for rhesus macaque iPSCs and could contribute to the advancement of preclinical studies using rhesus macaque iPSCs.

•PiggyBac transposon vector enables long-term gene expression in Rh-iPSCs.•Gene-transduced Rh-iPSCs form teratomas.•tEGFR-Rh-iPSCs maintain their expression after HSPC differentiation.•tEGFR-Rh-iPSCs maintained tEGFR expression after differentiation into T cells.•PiggyBac transposon vector as a simple method for gene transfection into Rh-iPSCs.

PiggyBac transposon vector enables long-term gene expression in Rh-iPSCs.

Gene-transduced Rh-iPSCs form teratomas.

tEGFR-Rh-iPSCs maintain their expression after HSPC differentiation.

tEGFR-Rh-iPSCs maintained tEGFR expression after differentiation into T cells.

PiggyBac transposon vector as a simple method for gene transfection into Rh-iPSCs.