A major collaborative effort that brought together researchers from the Institutes for Integrated Cell-Material Sciences (iCeMS) and Frontier Medical Sciences in Kyoto University (Kyoto, Japan), the Institute for Stem Cell Biology and Regenerative Medicine (inStem) and the National Centre for Biological Sciences (NCBS) (Bangalore, India), led to the development of a novel low-cost growth-factor-free culture medium that supports long term propagation of human pluripotent stem cells (hPSCs) derived either from human dermal fibroblast or peripheral blood mononuclear cells, published in Nature Biomedical Engineering this month.
Self-renewal and the potential to differentiate into all major lineages make hPCs excellent tools in regenerative medicine research and transplantation therapies; however, to date, production of xenobiotic-free medium requires the use of expensive recombinant human proteins, such as transforming growth factor-I? (TGF-I?) and high-dose basic fibroblast growth factor (bFGF), which along insulin and transferrin, are part of the simplest medium commercially available, Essential 8 (E8). The high costs associated with the aforementioned components, in addition to lot-to-lot variability, are major barriers to scaling hPCA production.
Previous work from the same group, led by Koichi Hasegawa, showed that Wnt signalling, which alone promotes hPCs proliferation, was suitable to support hPCs self-renewal and proliferation when coupled with ID-8, a chemical inhibitor of tyrosine phosphorylation-regulated kinase, DYRK); although this medium was free of bFGF and TGF-I?, it was still dependent upon expensive quality-controlled recombinant Wnt protein (Wnt3a). In order to replace Wnt3a (and the costs associated with it), the authors screened for inhibitors of glycogen synthase kinase 3I? (GSK3I?) inhibitors involved in the canonical Wnt/I?-catenin signalling pathway using KhEs-1 and H9 human embryonic cells (hECs). From all the screened small-molecule inhibitors of GSK3-I? (6-bromoindirubin-3a ?-oxime, CHIR99021, kenpaullone and 1-azakenpaullone), only a combination of 1-azakenpaullone plus ID-8 (AKI condition) proved to maintain long-term hPCs pluripotency, however, the proliferation rate of hPCs in this medium was lower than one observed in bFGF/TGF-I?-dependent culture systems. To compensate for this, the researchers supplemented the medium with tacrolimus, an inhibitor of nuclear factor activated T cells -cytoplasmic 1 (NFATc1), which expression was found to be increased in response to ID-8. Supplementation with tacrolimus (AKIT condition) was associated with large colonies of undifferentiated hPCs and doubled the expansion rate seen with AKI alone.
This novel chemically defined and growth-factor-free medium showed to be compatible with synthetic material that favors reproducible hPCs cultures, such as Synthemax, in addition to maintaining pluripotency over 30 passages, while also preserving genomic integrity, evaluated using comparative copy number variation and loss of heterozygosity analyses; however, expression profiles from hPCs cultured in AKIT and common culture systems had significant differences in gene expression profiles, which were attributed to variable regulation of the expressed genes. Further studies will be needed in order to assess the impact and mechanisms by which AKIT regulates metabolic states in hPCs, and to test if cells grown in this condition are capable to differentiate into relevant therapeutic cell lineages for biomedical research and applications.
The development of this chemically defined and growth-factor-medium will contribute to promoting large-scale hPCs production and their use in research, cell therapy, and drug discovery while maintaining lower costs and lower variability than those observed with current culture systems.