In this recently revised and expanded paper from our collaborators in the Bertero lab, Truszkowski et al. have described a cost-effective homemade media recipe for the culture of human pluripotent stem cells (hPSCs), taken with our recent application note [Commercial versus in-house media: a comparative study of human induced pluripotent stem cell maintenance] this adds to the evidence that homemade media using Qkine growth factors can be a suitable replacement for, or improvement on, commercially available media. The high cost of commercially available proprietary media and lack of transparency and flexibility can be a problem for academic labs and translational labs looking at scaling iPSC protocols.
Optimization of B8+ weekend-free media formulation
Maintenance of pluripotency in hPSCs relies on signaling through the activin/Nodal/TGF-β-SMAD2/3 and FGF2-MAPK pathways. FGF-2 is relatively unstable in cell culture conditions, leading to the need for daily media changes. Qkine thermostable FGF-2, FGF2-G3 is a modified, thermostable version of FGF-2 which remain stable and bioactive for >7 days under cell culture conditions. In this study, after validation of the thermostability and bioactivity of FGF2-G3 154 aa, the full length form and FGF2-G3 145 aa, a truncated form, FGF2-G3 145 aa was found to be more bioactive at lower concentrations. In fact, they found that the recommended concentration of 40 ng/ml FGF-2 was too high when using Qkine tag-free FGF2-G3 145 aa and negatively affected pluripotency. They found the optimal concentration of FGF2-G3 145 aa was 5 ng/ml, reducing the cost of FGF-2 by 8-fold overall. For activation of the activin/Nodal/TGF-β-SMAD2/3 they found activin A to be unsuitable and although TGF-β3 required a higher concentration than published methods, 1 ng/ml, TGF-β1 was most effective at an higher 2 ng/ml.
Validation of pluripotency with B8+
Once B8+ formulation had been optimized it was compared to a homemade E8 media (with 2 ng/ml TGF-β1) and a commercial E8 media. All media supported NANOG+/OCT4+ cell but iPSC cultured in B8+ media showed the highest NANOG expression. RNA sequencing allowed them to identify specific subpopulations of iPSC, those growth in weekend-free homemade media had some metabolic differences compared to commercial E8. Most notably the iPSC in B8+ were biased towards the neuroectoderm lineage. Differentiating iPSCs into neuroectodermal cells, followed by further differentiating into ectodermal lineages, is critical for studying neurodevelopmental processes, modeling neurological disorders, and developing potential therapies for conditions impacting the nervous system and skin [See application note].
Validation of iPSCs cultured in B8+ for downstream applications and differentiation
In order to test the suitability of iPSC for typical applications Truszkowski et al. compared iPSC in the different media in multiple downstream processes. They found that B8+ cultured cells performed best in single cell cloning and gene editing experiments. All lines were successfully differentiated into the three germ layers [see application notes below for our protocols]. More complex differentiations were also tested, differentiating into cardiac and colon organoids. Finally, a variety of iPSC lines across multiple laboratories were tested, to show that maintenance and differentiation of iPSC in the homemade media recipe was robust and reproducible.
All data summarized from Truszkowski et al. full data and media recipes available in the full publication.
Related Qkine application notes:
Differentiation of induced pluripotent stem cells (iPSCs) into endoderm
Differentiation of induced pluripotent stem cells (iPSCs) into mesoderm
Differentiation of induced pluripotent stem cells (iPSCs) into neuroectoderm