Cell-to-cell variability and culture conditions during self-renewal reversibly affect subsequent differentiation of mouse embryonic stem cells

Abstract

Cell-to-cell variability in clonal populations is reflected in a distribution of mRNA and protein levels among individual cells, including those of key transcription factors governing embryonic stem cell (ESC) pluripotency and differentiation. This may be a source of heterogeneity resulting in mixtures of cell types in differentiated populations despite efforts to control the differentiation conditions and the use of a clonal starting population. In addition, this distribution may be affected by the cell microenvironment during self-renewal. Prior studies on self-renewal culture of ESC, however, focused on long term proliferation and pluripotency. The effects of culture conditions during self-renewal on the effectiveness of subsequent differentiation protocols remains understudied. Using a mouse ESC line harboring a GFP reporter, we examined cell-to-cell variability in clonal undifferentiated populations and how such variability affects subsequent differentiation. Subpopulations sorted according to their levels of Oct4-GFP expression displayed distinctly different expression levels of pluripotency and early differentiation markers and differentiated into cardiomyocytes at different efficiencies. However, when allowed to self-renew after sorting, the subpopulations regenerated the parental distributions of Oct4-GFP and subsequent differentiation after regeneration did not show differences. In addition to differences between cells in a clonal population, self-renewal conditions affecting Oct4 expression on the population-level were examined. Changes in culture conditions during self-renewal by low oxygen culture or small molecule dual inhibition (2i) of mitogen-activated protein kinase and glycogen synthase kinase reversibly affected levels of Oct4 expression in cells that were otherwise pluripotent. Effects of different self-renewal conditions immediately preceding differentiation are manifested by changes in subsequent differentiation to cardiomyocytes. This study demonstrates that manipulation of self-renewal culture conditions can lead to changes in the outcomes of defined differentiation protocols, a novel dimension to explore for directed differentiation of pluripotent stem cells.