Cell-to-cell variability and culture conditions during self-renewal reversibly affect subsequent differentiation of mouse embryonic stem cells
Author(s)Tan, Jit Hin
Massachusetts Institute of Technology. Department of Chemical Engineering.
Clark K. Colton.
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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.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references (p. 133-151).
DepartmentMassachusetts Institute of Technology. Department of Chemical Engineering.; Massachusetts Institute of Technology. Department of Chemical Engineering
Massachusetts Institute of Technology