Objective: ‘Cultured meat’ has been suggested as means of solving the problems associated with overpopulation and gas emissions. Satellite cells are a major component in the production of cultured meat; however, these cells cannot be maintained in vitro over long periods. Fibronectin is a glycoprotein that affects biological processes such as cell adhesion, differentiation, and migration. Unfortunately, the characteristics of porcine satellite cells grown in a long-term culture when exposed to fibronectin-coated dishes are unknown. The objective of this study was to investigate the appropriate concentration of fibronectin coated dishes for proliferation and maintenance of porcine satellite cells at long-term culture.Methods: In this study, we isolated the satellite cells and fibroblast cells with pre-plating method. We next analyzed the cell doubling time, cell cycle, and rate of expressed paired box 7 (Pax7) and myogenic differentiation 1 (MyoD1) in porcine satellite cells cultured with 20 μg/mL of fibronectin-, gelatin-, and non-coated dishes at early and late passage. We then analyzed the proliferation of porcine satellite cells with various concentrations of mixed gelatin/fibronectin. We next determined the optimal concentration of fibronectin that would encourage proliferation and maintenance of porcine satellite cells in a long-term culture.Results: Doubling time was lowest when 20 μg/mL of fibronectin was used (as tested during an early and late passage). Levels of expressed Pax7 and MyoD1, assessed using immunocytochemistry, were highest in cells grown using fibronectin-coated dishes. The proliferation of gelatin/fibronectin mixed coatings had no significant effect on porcine satellite cells. The concentration of 5 μg/mL fibronectin coated dishes showed the lowest doubling time and maintained expression of Pax7.Conclusion: Fibronectin with 5μg/mL effectively maintains porcine satellite cells, a discovery that will be of interest to those developing the next generation of artificial meats.

Cultured meat produced using satellite cells has emerged to address issues such as overpopulation, the ethical conundrums associated with the breeding environment, and the methane gas emissions associated with factory farming. To date, however, the challenges of maintaining satellite cells in vitro and reducing the costs of the culture media are still substantial. Gelatin, collagen, and fibronectin are commonly used extracellular matrices (ECMs) that facilitate signal integration with the cells and promote cell adhesion. In this study, we compared the proliferation, cell cycle, immunocytochemistry, and expression levels of Pax7, Pax3, Myf5, MyoD1, and MyoG genes in bovine satellite cells (BSCs) cultured on gelatin-, collagen- and fibronectin-coated dishes as part of short- and long-term cultures. We observed that BSCs cultured on gelatin-coated dishes showed higher levels of Pax7 expression than BSCs cultured on collagen- and fibronectin-coated dishes in both short- and long-term cultures, indicating that BSCs cultured on gelatin effectively maintained the satellite cell population in both the short- and long-term cultures. Our study highlights that gelatin is an effective ECM for the maintenance of BSCs and the production of cultured meat.

Researching the technology for in vitro differentiation of embryonic stem cells (ESCs) into neural lineages is very important in developmental biology, regenerative medicine, and cell therapy. Thus, studies on in vitro differentiation of ESCs into neural lineages by co-culture are expected to improve our understanding of this process. A co-culture system has long been used to study interactions between cell populations, improve culture efficiency, and establish synthetic interactions between populations. In this study, we investigated the effect of a co-culture of ESCs with neural stem cells (NSCs) in two-dimensional (2D) or three-dimensional (3D) culture conditions. Furthermore, we examined the effect of an NSC-derived conditioned medium (CM) on ESC differentiation. OG2-ESCs lost the specific morphology of colonies and Oct4-GFP when co-cultured with NSC. Additionally, real-time PCR analysis showed that ESCs co-cultured with NSCs expressed higher levels of ectoderm markers Pax6 and Sox1 under both co-culture conditions. However, the differentiation efficiency of CM was lower than that of the non-conditioned medium. Collectively, our results show that co-culture with NSCs promotes the differentiation of ESCs into the ectoderm.

Mouse pluripotent stem cells are generally classified into two interchangeable states: naïve pluripotency and primed pluripotency, are represented by embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), respectively (Nichols & Smith, 2009). ESCs are derived from the inner cell mass (ICM) of blastocyst-stage embryos and are characterized by a dome-shape, dense cluster morphology (Boroviak et al., 2014; Martello & Smith, 2014). In contrast, EpiSCs are derived from epiblast at E5.5-7.5 after implantation and exhibit a flat, monolayer-spreading cluster morphology (Najm et al., 2011; Kojima et al., 2014). Although both refer to pluripotency, they significantly differ in various biological characteristics, including morphology, gene expression patterns, maintenance conditions, epigenetic status, chimerism contribution ability, and germ cell differentiation ability (Tsukiyama & Ohinata, 2014; Yu et al., 2021). In particular, EpiSCs have been reported to resistant to differentiation into primordial germ cell-like cells (PGCLCs) (Hayashi et al., 2011), suggesting that EpiSCs are already in a developmentally biased state. In comparison, epiblast-like cells (EpiLCs) mimic the state of the upper germ layer at E5.75 in vivo and are highly responsive to PGC-inducing cytokines including bone morphogenetic protein 4 (BMP4), allowing them to effectively differentiate into PGCLCs (Hayashi et al., 2011; Nakaki et al., 2013). Although both EpiSCs and EpiLCs belong to a primed pluripotent state, EpiLCs are known to have a higher reprogramming ability and responsiveness to PGCLC induction because they mimic a narrower developmental time point reflecting the epithelial epiblast before gastrulation (Endoh & Niwa, 2022).