Toward exploring cellular metastates through light-induced tuning of Oct4 Expression

Abstract

Embryonic stem cell (ESC) and Epiblast stem cell (EpiSC) are widely used to understand embryogenesis process. Researchers use in vitro differentiation of ESC or EpiSC to mimic in vivo development, to understand Gene Regulatory Network regulating development, and to generate functional terminal differentiated cells. The approaches include adding chemicals or biomolecules to activate intracellular signal pathways or modify cell microenvironment, or to directly modify transcriptional factor expressions. Cells expressing markers for hematopoietic cells, hepatocytes, germ cells, kidney and cardiac cells have been successfully generated in previous studies. However, two obstacles prevent clinical applications of these in vitro differentiated cells. One is cell identity heterogeneity and the other is low in vitro differentiation efficiency. Cell identity heterogeneity raises safety issue in clinical application, and low differentiation efficiency results in high cost for clinical application.

To help understand and visualize development process, Conrad Hal Waddington, and Jin Wang & Sui Huang proposed ‘epigenetic landscape’ and ‘pseudo-potential landscape’ respectively. Waddington’s epigenetic landscape shows the bifurcations during differentiation from stem cells to terminal differentiated cells, but it is hard to analyze landscape quantitatively. Jin Wang & Sui Huang introduced concepts in statistical physics, and tried to quantify Waddington’s epigenetic landscape by the probability of observation of cells at different cellular states in phase space defined with quantities of transcriptional factors. Some are called cellular metastates or attractors, as they represent stable cell status in the phase space. The existence of cellular metastates or attractors is a product of Gene Regulatory Network, and potential landscape could help analyze transition between these states theoretically. However, there has been no study that explores relations between transcriptional factors’ quantities and cellular metastates experimentally. In this study, we quantitatively tuned Oct4 expression in cells through specifically knock down endogenous Oct4 using doxycycline-inducible miRNA30 based RNAi and induced exogenous Oct4 expression using light-induced expression system. We verified the existence of cellular metastate experimentally and found four metastates besides the ES state. After transcriptome analysis, the four metastates are related to robust pluripotency, trophectoderm or endoderm, mesendoderm and mesoderm, respectively. We also discover an unknown regulation that downregulated Oct4 expression after losing Nanog-GFP transcription. We would further verify the metastates’ identifications by comparing their differentiation efficiencies to predicted lineages with ES cells, using standard in vitro differentiation protocols. Our study emphasizes the importance of quantitative, time lapse analysis at single cell level when studying dynamic and quantity-sensitive process such as cell differentiation. In the future, we will integrate experimental platform developed in this study, and advanced real-time feedback control algorithms for precise control of exogenous transcriptional factors. We believe that, with this quantitative approach, we will be able to generate pure terminate differentiated cells for safe clinical applications.