Thermostable (heat stable)

bFGF / FGF2-G3

Heat stable FGF-2 for stem cell culture B8 media

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

Wild-type FGF-2 (bFGF)

FGF-2 / bFGF 145 aa (Qk025)
FGF-2 / bFGF 154 aa (Qk027)

Thermostable (heat stable) FGF2-G3

Thermostable bFGF / FGF2-G3  / FGF2-STAB ® 145 aa (Qk052)
Thermostable bFGF / FGF2-G3 154 aa (Qk053)

Other FGF-2 (bFGF)

FGF-2 / bFGF (Qk002)
mouse FGF-2 (Qk042)

Key papers

Dvorak et al. 2018

development of FGF2-G3 form using AI

Koledova et al. 2019

assessment of FGF2-G3 stability and signalling

Kuo et al. 2020

development of B8 media – low cost, weekend free iPSC media

Lyra-Leite et al. 2021

updated protocol for B8 media

Benington et al. 2020

nice review of FGF2 stability and stabilisation methods

download poster

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

Koledova and colleagues at Masaryk University explored the receptor specificity of FGF2-G3 (FGF2 STAB®) in detail using a BaF3 cell proliferation assay. FGF2-G3 induced proliferation of cells expressing FGFR1c, FGFR2c, FGFR3c and FGFR1b and not FGFR2b and FGFR3b, the same profile of receptor activation as wild-type FGF-2. Interestingly they also report a lower EC50 value (10-100x lower) for FGF2-G3 than wild-type and suggested the stability increased receptor affinity in this assay.
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
Petr Dvorak and Jiri Damborsky, researchers at Masaryk University, published their computer-assisted protein engineering approach, used to generate forms of FGF-2 with greater thermal and chemical stability, improving the half-life at 37 oC from 10h to more than 20 days. In a multi-step process, Dvorak and colleagues identified an optimal set of nine amino acid substitutions that stabilized FGF-2 whilst not affecting the FGF receptor 1 (FGFR1) or FGF receptor 2 (FGFR2) interface. This hyperstable FGF2 is known as FGF2-G3, or FGF2-STAB® (Dvorak et al. 2017). The biological half-life of FGF2-G3 and previous stabilized FGF2 iterations (FGF2-G0 and FGF2-G2) were evaluated by phosphorylation of ERK1/2 through FGF2 receptor signalling in FGF-2 starved human embryonic stem cells. In these assays, FGF-2 was incubated in conditioned hESC media prior to the assay to mimic time in culture. The biological activity of wild-type FGF2 was <50% after 10h incubation with conditioned media. In contrast, no significant reduction in FGF2-G3 biological activity was observed after up to 20 days of incubation with media at 37oC. In the same study, FGF2-G3 and wild-type FGF-2 had equivalent ability to maintain hESC pluripotency and expression of pluripotency markers Oct-4 and nanog.
Patent (WO2017089016A1) filed describing the FGF2-G3 technology by Masaryk University and Enantis and its potential for regenerative medicine and stem cell culture applications.
E8 media, developed by Chen and colleagues (Chen et al. 2011, Beers et al. 2012), was adopted as a standard base media for human induced pluripotent stem cell culture. This media provided a xeno-free, chemically defined stem cell medium with FGF-2 and a TGF beta protein supporting maintenance of pluripotency. Many variations of E8 have been optimized for specific iPSC cell lines and applications.

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.

1985 FGF-2 (first called basic FGF, bFGF) was isolated as a potent mitogen 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.

Resources

Why should I use animal-free growth factors and cytokines?
The future of iPSC-derived pancreas and liver implants
BMP4 comparison with other suppliers: Qkine BMP4 is more potent
BMP-4 (Qk038) has enhanced bioactivity

FGF2-G3 stem cell applications

Whilst B8 is the first media using FGF2-G3, there is enormous potential for a step-change in stem cell culture protocols.

Try it.  Be part of the revolution!

Thermostable FGF 2 G3 for iPSC stem cell media

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