Recombinant production of canine vitronectin for optimizing the culture of canine induced pluripotent stem cells

Abstract

Introduction

Canine induced pluripotent stem cells (ciPSCs) have attracted attention as valuable tools in veterinary regenerative medicine and disease modeling. Feeder-free culture of ciPSCs using iMatrix-511 has become feasible. Since the choice of extracellular matrix (ECM) has been shown to significantly affect not only the maintenance of pluripotency but also the efficiency of directed differentiation, systematic evaluation of multiple ECM substrates is considered important in ciPSC culture as well. Furthermore, considering future clinical applications, it is essential to establish a xeno-free culture system. Vitronectin (VTN) is a protein that can be easily expressed as a recombinant product in Escherichia coli, making it suitable for scalable ECM production. In this study, we generated recombinant canine-derived VTN and evaluated its effects on ciPSCs in comparison with human-derived ECM substrates.

Methods

In this study, we generated recombinant full-length and N-terminally truncated forms of canine vitronectin (cVTN and cVTN-N) using a bacterial expression system. These substrates, along with established human-derived ECM proteins including iMatrix-511, hVTN, and hVTN-N, were evaluated for their ability to support ciPSC adhesion, proliferation, and the maintenance of pluripotency and differentiation potential. Pluripotency and differentiation capacity were assessed using immunostaining and gene expression analysis.

Results

Both cVTN and cVTN-N demonstrated support for ciPSC attachment and long-term proliferation at levels comparable to those of human-derived ECM substrates. ciPSCs cultured on cVTN or cVTN-N maintained high expression levels of pluripotency markers; in particular, cVTN significantly enhanced SOX2 expression, while cVTN-N was associated with reduced mesodermal marker expression. Efficient EBs formation and trilineage differentiation were achieved on all tested substrates, with only minor differences in lineage marker expression among groups.

Conclusions

Recombinant canine-derived VTN were shown to function as an effective, species-matched substrate for ciPSC culture, exhibiting comparable performance to human-derived ECM proteins. These results suggest that canine-derived VTN enables stable proliferation and maintenance of pluripotency in ciPSCs, providing a promising platform for future research in canine regenerative medicine.