# Wisent Somatic Cells Resist Reprogramming by the PiggyBac Transposon System: A Case Study Highlighting Methodological and Conservation Hurdles

## TL;DR

This study explores the difficulty of reprogramming wisent somatic cells into stem cells, highlighting challenges in conservation and the need for new methods.

## Contribution

The study reveals intrinsic barriers to reprogramming wisent cells and suggests the need for alternative strategies like new delivery systems.

## Key findings

- Wisent somatic cells showed limited proliferation and unstable pluripotency after reprogramming.
- Optimized culture conditions failed to significantly improve reprogramming efficiency.
- Alternative strategies like new-generation delivery systems may be needed for successful reprogramming.

## Abstract

The European wisent (Bison bonasus), an iconic yet genetically vulnerable species, faces ongoing conservation challenges due to a restricted gene pool. Advances in induced pluripotent stem cell (iPSC) technology offer promising prospects for preserving and restoring genetic diversity in endangered species. In this study, we sought to reprogram wisent somatic cells into iPSCs using the PiggyBac transposon system, a non-viral method known for being successfully applied in bovine species. We applied a six-factor reprogramming cocktail (OCT4, SOX2, KLF4, LIN28, c-MYC, NANOG) alongside small-molecule enhancers to fibroblasts isolated from adult wisent tissue. While initial colony formation was observed, the reprogrammed cells exhibited limited proliferation and failed to maintain stable pluripotency, suggesting intrinsic barriers to complete reprogramming. Despite optimizing culture conditions, including hypoxia and extracellular matrix modifications, the reprogramming efficiency remained low. Our findings indicate that wisent somatic cells may require alternative reprogramming strategies, such as new-generation delivery systems and epigenetic modulators, to achieve stable iPSC lines. This study underscores the need for species-specific optimization of reprogramming protocols and highlights the potential of emerging cellular technologies for conservation efforts. Future research integrating advanced reprogramming tools may pave the way for genetic rescue strategies in wisent and other endangered species.