FGF-2 (also known as basic FGF or bFGF) is an essential growth factor for maintaining human embryonic stem cell (hESC) and induced pluripotency stem cell (iPSC) pluripotency in feeder-free and chemically defined stem cell media. It is a core component of widely adopted media including mTESR (Ludwig et al. 2006), StemPRO (Wang et al. 2007) and E8 (Chen et al. 2011). However, FGF-2 is inherently unstable and prone to proteolytic degradation and aggregation. This fundamental biochemical instability, and therefore low half-life in culture media (<10 h), is an important contribution to the need for frequent media changes and challenges in improving homogeneity during stem cell proliferation and subsequent differentiation.
To improve the stability of FGF-2 for stem cell media and regenerative medicine applications, Dvorak and colleagues at Masaryk University used computer-assisted protein engineering to identify an optimal set of nine amino acid substitutions that stabilize FGF-2. These substitutions were designed to avoid structural changes to the FGF receptor 1 (FGFR1) and FGF receptor 2 (FGFR2) binding sites. This thermostable FGF-2 is known as FGF2-G3, or FGF2-STAB® (Dvorak et al. 2017). The biological activity of wild-type FGF-2 is <50% after 10h incubation with conditioned media. In contrast, no reduction in FGF2-G3 biological activity is observed after >7 days incubation with conditioned media at 37oC. Both FGF2-G3 and wild-type FGF-2 maintain hESC pluripotency and expression of pluripotency markers Oct-4 and nanog with equivalent efficacy (Dvorak et al. 2017).
In 2020, Paul Burridge and colleagues at Northwestern University, Chicago, published a protocol for B8 media. This iPSC maintenance media uses thermostable FGF2-G3, along with optimization of media component concentration and composition to reduce media cost and facilitate weekend-free stem cell culture regimes. (Kuo et al. 2020 and updated in Lyra-Leite et al. 2020).
To manufacture and provide FGF2-G3 for stem cell culture, including use in B8 media and emerging applications such as cultured meat, Qkine has licensed the patented FGF2-G3 technology from Enantis/Masaryk University. We have combined the excellent science behind the FGF2-G3 technology with our protein manufacture expertise to provide gold standard protein for use in cell culture media. We have removed His tags present in academic forms of the protein, as these may give rise to issues for scientists translating discoveries to the clinical or scale-up. His tags also introduce unnecessary scientific uncertainty.