Rising, Anna
- Department of Animal Biosciences, Swedish University of Agricultural Sciences
- Karolinska Institute
Conference abstract2015
Rising, Anna; Andersson, Marlene; Kronqvist, Nina; Chen, Gefei; Wu, Siqin; Otikovs, Martins; Westermark, Per; Hovatta, Outi; Chesler, Mitchel; Jaudzems, Kristaps; Johansson, Jan
There is a great need for defined cell culture systems that allow expansion of human pluripotent stem cells (hPSCs) and subsequent controlled differentiation, ideally in an implantable three-dimensional (3D) matrix. Spider silk appears to be an ideal biomaterial, since it is strong, extendible, and has favorable properties when implanted in living tissues. Spiders are difficult to house and therefor methods for recombinant production of spider silk are warranted. We have developed a method for production of recombinant spider silk fibers, films and foams that are used for the design of defined and xeno-free cell culture matrices. The matrices enable long-term expansion of multiple hPSC lines and subsequent differentiation into all three germ layers in 3D. This hPSC culture method provides robust, defined, easily produced and flexible culture environments for hPSCs (1). These matrices are promising but to realize their full potential, we need to spin continuous fibers in a reproducible way. Spider silk fibers are produced from soluble spidroins under ambient conditions. The spidroins are large and highly repetitive in sequence but capped by non-repetitive N- and C-terminal domains (NT and CT). In the gland, a pH gradient, that goes from 7.6 to <5.7, is generated by active carbonic anhydrase. The terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, while CT on the other hand is destabilized and unfolds into b-sheet amyloid fibrils, which can trigger fiber formation (2,3). There is a high pCO2 in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by NMR spectroscopy. These simultaneous events constitute a novel CO2 and proton dependent lock and trigger mechanism of spider silk formation that possibly can be harnessed in biomimetic spinning of artificial spider silk.
1. Wu S, Johansson J, Damdimopoulou P, Shahsavani M, Falk A, Hovatta O, Rising A. Spider silk for xeno-free long-term self-renewal and differentiation of human pluripotent stem cells. Biomaterials. 2014 Oct;35(30):8496-502.
2. Kronqvist, N., Otikovs, M., Chmyrov, V., Chen, G., Andersson M., Nordling, K., Landreh, M., Sarr, M., Jörnvall, H, Wennmalm, S., Widengren, J., Meng, Q., Rising, A., Otzen, D., Knight, S. D., Jaudzems, K., Johansson, J. Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation Nat Comm. 2014. 10(5):3254.
3. Andersson M, Chen G, Otikovs M, Landreh M, Nordling K, Kronqvist N, Westermark P, Jörnvall H, Knight S, Ridderstråle Y, Holm L, Meng Q, Jaudzems K, Chesler M, Johansson J, Rising A. Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains. PLoS Biol. 2014 Aug 5;12(8):e1001921
Publisher: MRS (Materials Research Society)
2015 MRS Spring Meeting. San Francisco, California. April 6-10
Cell and Molecular Biology
https://res.slu.se/id/publ/68656