Effects of Essential 8 (E8) media additives polyethylene glycol (PEG) and Heparin sodium salt (HS) on the pluripotency and oncogenes

Manuscript Name: "Addressing Bioreactor hiPSC Aggregate Stability, Maintenance and Scaleup Challenges Using a Design of Experiment Approach" Evaluation of core pluripotent genes showed that expression levels were similar between samples with a clear indication of a primed phenotype. In addition, we evaluated several well-known oncogenes notable TP53, MYC, NANOGP8, EEF1A2 and KLF4 were expressed with similar transcript levels between samples, though KLF4 had low overall expression. It was observed that a few genes indicative of forward differentiation were expressed with TSR genes TDGF1, LEFTY1 and IL17RC and the trophectodermal genes KRT8 and TEAD4 showing comparable expression levels. Expression of the primary primitive streak gene NODAL was observed at low transcript levels in both samples. Examining the top differentially expressed genes we noted that there were several genes involved in ectodermal differentiation and some key signaling pathway components that were significantly downregulated in response to the presence of PEG and HS. Down-regulated ectodermal genes included SOX1, OLIG3, LHX5 and OTX2 as well as some less known ones. The pathways most downregulated were the TGFβ family as suggested by BMP2 and BMP4 down regulation, though the previously mentioned levels of TDGF1, LEFTY1 and NODAL remained consistent to cultures lacking additives. Wnt family signaling components down regulated were Wnt4, FRZB, FZD5 and FRZ8. Also a decreased in retinoic acid signaling components were noted as indicated by decreased CYP26A1, CYP26C1, DHRS3 and CRABP2 transcript levels.