Pluto Bioinformatics

GSE84137: Vitamin C and L-Proline antagonistic effects capture alternative states in the pluripotency continuum [RNA-Seq]

Bulk RNA sequencing

Samples 1-4 report RNA-seq transcriptome profiling of the L-Proline- (L-Pro) and bFgf/ActivinA- (F/A) derived mCherry+/eGFP+ (yellow) ESC population, using the Illumina HiSeq platform. Whole-genome expression revealed that more than 1000 genes were significantly deregulated in L-Pro- and F/A-induced cells compared to control (mCherry+/eGFP red cells) and the two population shared up to 75% of deregulated genes with the same deregulation trend. Specifically, the pluripotency-associated genes were downregulated either at similar level (Nanog, Klf2, Klf4 and Gbx2) or at lower levels (up to 10 times) (Dppa 2, 3, 4, 5a, Rex1, Esrrb) in F/A- compared to L-Pro-treated cells. Interestingly, mesendodermal-related genes (e.g. Brachyury, Cer1, Dkk1, Eomes, Foxa2, and Sox17) were induced in both conditions but at significant higher levels in F/A- compared to L-Pro-treated cells. The transcriptome analysis of mCherry+/eGFP+ (yellow) cells supported the idea that L-Pro mimics F/A in inducing a nave to primed transition, and suggested that it exerted a milder (weaker) effect. Samples 5-14 report RNA-seq transcriptome profiling of the mir-290_mCherry/mir-302_eGFP dual reporter ESCs (DRESCs) bulk culture, grown in FBS/LIF VitaminC (VitC) and L-Proline (L-Pro) and compared them to the standard naive/2i and primed/bFgf/ActivinA-EpiSCs (F/A), using the Illumina HiSeq platform. Whole-genome expression identified around 7900 deregulated genes in the different conditions, (fold change2 and pvalue<0.05). Principal component analysis (PCA) placed VitC between 2i and untreated control, and L-Pro between control and F/A. Accordingly, a set of pluripotency-associated genes was expressed at higher level in 2i and VitC conditions, while downregulated in L-Pro and F/A, compared to control. Conversely, priming markers were downregulated in 2i and VitC and upregulated in L-Pro and F/A compared to control The transcriptome analysis supported that VitC- and L-Pro captured alternative pluripotency states that can be likely placed between nave/2i and primed/F/A states. SOURCE: Cristina D'Aniello (cristina.daniello@igb.cnr.it) - IGB-CNR