In vitro overexpression of Nhp2, Emg1 and Tsr3 genes in iPSC-derived neural crest cells

During embryonic development, the neural crest contributes to craniofacial formation by differentiating into skeletogenic mesenchyme and neuro-glial lineages. Previous single-cell transcriptomic analysis revealed that mesenchymal fate commitment correlates with expression of rRNA-modifying and ribosome assembly factors, including EMG1 and NHP2, which introduce post-transcriptional modifications into 18S rRNA (notably m¹acp³? at U1248) with final maturation requiring TSR3. To explore the functional importance of m¹acp³? modification in the embryonic neural crest, we utilized lentiviral vectors to overexpress of Nhp2, Emg1, and Tsr3 in iPSC-derived neural crest cells. Overall design: Human iPSCs were infected with lentiviral vectors carriyng the exogeneous Nhp2, Emg1 or Tsr3, and then were differentiated into neural crest cells, with doxycycline-induced overexpression of NHP2, EMG1, or TSR3 during differentiation; cells were collected at day 6 and analyzed by 10x Genomics Chromium Single Cell 3' RNA-seq.