Highly optimized stem cell supplement for weekend-free cell maintenance
HiDef-B8 Stem Cell Medium Supplement is the formulation of a hiPSC culture medium that resulted from an exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. The eight key ingredients are Insulin, Ascorbic acid 2-phosphate, Transferrin, Sodium selenite, FGF2-G3, TGFB3, NRG1, and Sodium bicarbonate. A recent publication from Northwestern University demonstrates the derivation and culture of 34 hiPSC lines in HiDef-B8 as well as the maintenance of pluripotency long term (over 100 passages). This formula also allows a weekend-free feeding schedule without sacrificing capacity for differentiation.
QUALIFICATION OF HIDEF-B8
The gold-standard demonstration of the suitability of a hiPSC medium is the capacity to generate hiPSC lines and maintain them in long-term culture. Our standard hiPSC line 19c3 has been cultured for >100 passages (p) in HiDEF-B8 and maintained expression of markers of undifferentiated status (SSEA4 and TRA-1-60) by flow cytometry (p131 at time of assay). We also generated hiPSC lines from 37 patients using established protocols but using B8. These hiPSC lines maintained SSEA4 and TRA-1-60 expression in culture (up to p65 at time of assay). In B8 hiPSCs maintained an hESC-like morphology; positive immunofluorescent staining for SSEA4, POU5F1, SOX2, and TRA-1-60; and normal karyotype.
Demonstration of the expression of markers of undifferentiated status in hiPSC line 19c3 cultured in B8 from passage (p) 21 to p131 (left) and 37 individual hiPSC lines derived in B8 from p12 to p65 (right), assessed by flow cytometry.
Phase-contrast images (original magnification 103) of hiPSC line 19c3 p44 cultured in B8. Scale bar, 100 mm.
Expression of markers of undifferentiated status in a variety of B8-derived hiPSC lines. Scale bar, 100 mm.
Example G-banding karyotype analysis of four hiPSC lines derived in B8.