Generation of bovine iPSCs from fetal fibroblasts for in vitro myogenesis and cultured meat

Kaiana Recchia¹Methi Wathikthinnakon²Fabiana Fernandes Bressan¹Kristine Freude^2*

• ¹Departament of Surgery, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, São Paulo, Brazil
• ²Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Capital Region of Denmark, Denmark

Emerging biotechnologies are increasingly explored in food production, such as cell-cultivated meat production. However, conventional methods rely on satellite cells (SCs) biopsies, which faces scalability challenges. Bovine induced pluripotent stem cells (biPSCs) offer a promising alternative due to their self-renewal and plasticity. This study employed both lentiviral (integrating) and episomal (non-integrating) reprogramming strategies to generate biPSCs for myogenic differentiation. Bovine fetal fibroblasts (bFFs) were reprogrammed using episomal vectors (pMaster K and pCXB-EBNA1), yielding putative colonies 13 days post-nucleofection. A selected clonal line (bFF-iPSCs pMK) expressed key pluripotency markers (AP, OCT4, SOX2, NANOG) and was stably maintained for over 33 passages, although episomal plasmids remained detectable. Comparative in vitro myogenesis of the bFF-iPSCs pMK and a previously established lentiviral line (bFF-iPSCs mOSKM) showed downregulation of OCT4, SOX2, and NANOG, with concurrent upregulation of the early myogenic marker PAX3. By day 30, the bFF-iPSCs pMK line exhibited elongated, multinucleated cells characteristic of myotubes, along with a corresponding gene expression profile. These findings offer new insights into bovine in vitro myogenesis and its potential for cultured meat applications. However, full myogenic differentiation remains challenging, emphasizing the need for further optimization.