FGF2-G3 is a hyperstable form FGF-2 (bFGF), the essential component for the maintenance of pluripotency in stem cell media.
FGF-2, also known as basic FGF or bFGF, is the essential component in all embryonic and induced pluripotent stem cell media for maintenance of pluripotency. 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.
We have licensed thermostable (heat stable) FGF-2 (bFGF) technology – FGF2-G3 – from Enantis. This innovation was combined with our manufacture expertise to generate an animal-free FGF2-G3 without protein to provide compatibility with stem cell culture process development and scale-up. Additionally, we offer both FGF2-G3 145aa (Qk052) and the 154aa original form (Qk053) so you can compare directly with the wild-type FGF-2 that you currently use. If you would like to evaluate FGF2-G3, please contact us.
FGF-2 (bFGF) proteins for media optimization
Key papers
A brief history of FGF-2 in stem cell culture
Qkine licensed the patented FGF2-G3 technology from Enantis/Masaryk University to manufacture and provide FGF2-G3 for stem cell culture and emerging applications such as cellular agriculture.
At Qkine, we have combined the excellent science behind the FGF2-G3 technology with our protein manufacture expertise to provide the best quality protein. As His tags may cause issues for scientists looking to translate discoveries to clinical or scale-up applications, we optimized the production of FGF2-G3 to remove His tags present in the academic constructs. Tags both are challenging to explain to regulatory bodies and introduce scientific uncertainties.
Two forms of FGF2 are used frequently in stem cell culture, the 146 aa (or 145 aa) form and a 154 aa form with a 9 residue extension at the N-terminus. The 9 aa pro-segment of FGF2 is not required for biological activity and may have roles in the localization of FGF2. The original FGF2-G3 (Qk053) corresponds to the 154 aa protein. We also wanted to allow scientists to compare directly with their existing FGF-2, so we introduced the nine amino acid substitutions into the 145 aa form of FGF2 to make Qk052 FGF2-G3 (145 aa).
Paul Burridge and colleagues at Northwestern University, Chicago, published a protocol for B8 media. This media uses thermostable FGF2-G3, along with optimization of media component concentration and composition to reduce media cost and facilitate weekend-free pluripotent stem cell culture regimes. B8 media effectively maintains the homogeneity and differentiation potential of human iPSC (Kuo et al. and updated in Lyra-Leite et al.).
The main changes in B8 media compared to the industry-standard E8 media are:
• replacing FGF-2 (100 ng/ml) with FGF2-G3 (used at 5 ng/ml)
• reduce pH to 7-7.1 and osmolarity to 310 osm/L
• reduce the concentration of TGF beta 1 (Qk010) to 2ng/ml
In the same study, the authors evaluated the impact of exogenous heparin on FGF-2 wild-type and FGF2-G3 bioactivity (EC50) and signalling dynamics. Heparin has a stabilising effect on WT FGF-2 and, perhaps consequently, impacts FGF2 signalling dynamics. FGF2-G3 signalling was less heparin-dependent in ERK1/2 signalling assay in both the FGFR over-expressing cell lines and in primary fibroblast
Ludwig and colleagues establish a feeder-free media for human embryonic stem cell culture (mTESR). FGF-2 is identified as the most important factor for maintaining hESC pluripotency in feeder-free media. The original publications use a zebrafish FGF2, although subsequent mTESR formulations use a human FGF2 protein. An alternative culture media, StemPro, which also uses FGF2 as a core component, was developed in parallel (Wang. 2007). hESC media are reviewed in Lee et al. 2011
The unusual initiation start sites and multiple isoforms of FGF2 were characterized. FGF2 has five alternative initiation sites, four of which are atypical CUG codons and one AUG. The product produced from the AUG site is the 18 kDa protein corresponding to the 154 aa form adopted for use in stem cell culture media. FGF2 does not contain a signal sequence and is secreted through the membrane using a mechanism dependent on the tertiary structure and exposed thiols. Interestingly, instead of a signal sequence, a 9aa N-terminal pro-segment is subject to proteolytic cleavage to form a 146 aa mature segment. This 146aa protein is the form original purified from tissues.
FGF-2, also known as basic FGF or bFGF, was isolated from several animal tissues as one of two main mitogenic proteins for endothelial cells (acidic and basic FGF). Esch et al determined the amino acid sequence of FGF-2 from bovine pituitary. This corresponds to the 146 aa form of FGF-2.
