Supporting data for Investigation on stem cell-associated transcription factors-artificial evolution of KLF4 and natural evolution of Sox

In the first study of KLF4, we aimed to optimize methods for iPSC generation by artificially evolving and enhancing the function of KLF4. We identified an engineered version of KLF4, referred to as eKLF4 by counting GFP-positive colonies to prove its improved function in iPSC reprogramming. Validation assays of Immunocytochemistry and qPCR were used to characterise the iPSCs generated by eKLF4. Mechanistic studies by collecting samples for RNA-seq and ATAC-seq and analyzed using Rstudio unveiled that eKLF4 drives a mesenchymal to epithelial transition at the early stages of reprogramming. It also opens up pluripotency-associated chromatin regions and activates the pluripotency network, thereby promoting iPSC generation. Overall, our study demonstrates that eKLF4, with its enhanced function compared to wildtype KLF4 for rapid and efficient reprogramming.

In the second project of uSOX, we discovered the emergence of two essential stemness-associated factors, SOX and POU, predates the evolution of animal multicellularity. By iPSC reprogramming tests by counting GFP-positive colonies for quantification, our study revealed that unicellular organisms possess POU and SOX sequences. Validation assays of Immunocytochemistry and qPCR were used to characterise the iPSCs generated by uSOX. Collectively, our findings shed new light on the evolutionary origins of stemness-associated factors.