Abstract
Reactive oxygen species (ROS) play important roles as second messengers in a wide array of cellular processes including differentiation of stem cells. We identified Nox4 as the major ROS-generating enzyme whose expression is induced during differentiation of embryoid body (EB) into cells of all three germ layers. The role of Nox4 was examined using induced pluripotent stem cells (iPSCs) generated from Nox4 knockout (Nox4−/−) mouse. Differentiation markers showed significantly reduced expression levels consistent with the importance of Nox4-generated ROS during this process. From transcriptomic analyses, we found insulin-like growth factor 2 (IGF2), a member of a gene family extensively involved in embryonic development, as one of the most down-regulated genes in Nox4−/− cells. Indeed, addition of IGF2 to culture partly restored the differentiation competence of Nox4−/− iPSCs. Our results reveal an important signaling axis mediated by ROS in control of crucial events during differentiation of pluripotent stem cells.Graphical
Highlights
The range of cellular processes Reactive oxygen species (ROS) mediates is continuously expanding and includes those associated with cell signaling and homeostasis [1,2,3]
Application of N-acetyl Cysteine (NAC) did not lead to reduction in the size of outgrowth suggesting that while activation of specific signaling by NADPH oxidase (Nox) enzyme or enzymes is required for the embryoid body (EB) outgrowth, the global removal of ROS mediated by NAC has other benefits for EB growth (Supplementary Fig. 1)
While terminally differentiated cells expressing specific markers were readily found in clusters in control cultures, the addition of Diphenyleneiodonium chloride (DPI) reduced the number of such cells
Summary
The range of cellular processes ROS mediates is continuously expanding and includes those associated with cell signaling and homeostasis [1,2,3]. Jusong Kim and Jaewon Kim contributed to this work. Stem cells are important tools for cell-based regenerative therapies. ROS are known to regulate self-renewal and differentiation of various stem cells including pluripotent embryonic stem (ES) cells [6, 8, 9]. Nox and Nox have been most frequently reported to function as the source for ROS during stem cell differentiation [10,11,12,13]. Most of the studies including those targeting ES cells and iPSCs
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