GSE137289
Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture
Purpose: To establish a molecular fingerprint for optoWnt-induced differentiation, we measured global transcriptional changes using bulk-population RNA-seq of optoWnt and WT hESCs after 48 hrs of light stimulation. Illuminated WT cells served as a phototoxicity control, and unilluminated optoWnt cells controlled for potential CRISPR/Cas9 knock-in effects, cell perturbation due to optoWnt expression, and dark-state Wnt pathway activation. We also measured feedback between optoWnt/WT populations during co-culture (1:1) by FACS sorting for each population after 48 hrs of growth. Methods: Bulk RNA-seq of hESCs using Illumina TruSeq Stranded mRNA Library Prep Kit and sequenced on HiSeq 4000 Results: Principal component analysis (PCA) showed clustering of biological triplicates for each condition and strong transcriptional changes upon optoWnt stimulation that account for 95% gene variance among samples. Dark and illuminated monoculture WT cells clustered together, and differential analysis of monocultures showed minimal gene expression differences, demonstrating minimal phototoxicity effects after 48 hrs of continuous 0.8 µW/mm-2 blue light stimulation. In contrast, optoWnt stimulation induced a broad transcriptional effect, with ~5,500 differentially expressed genes between the dark and illuminated conditions. Direct B-catenin target genes, such as CDX1, DKK1, T, and TBX3, are among the most differentially expressed genes, all with a log fold change (LFC) of ~ 9 – 13. Lastly, WT cells grown in illuminated co-culture with optoWnt cells showed differential gene expression and upregulation of TGF-B target genes when compared with WT cells grown in monoculture or in dark co-cultures, indicating signaling feedback between the optoWnt and WT populations. Overall design: Bulk RNA sequencing of WT and optoWnt hESCs in triplicate. Cells were cultured in monoculture or in optoWnt/WT co-cultures (1:1) and were FACS sorted for OptoWnt (mCherry+) and WT (GFP+) cells.