Abstract
Cellular organelles play fundamental roles in almost all cell behaviors. Mitochondria have been reported to be functionally linked to various biological processes, including reprogramming and pluripotency maintenance. However, very little about the role of mitochondria has been revealed in human early development and lineage specification. Here, we reported the characteristics and function of mitochondria during human definitive endoderm differentiation. Using a well-established differentiation system, we first investigated the change of mitochondrial morphology by comparing undifferentiated pluripotent stem cells, the intermediate mesendoderm cells, and differentiated endoderm cells, and found that mitochondria were gradually elongated and matured along differentiation. We further analyzed the expression pattern of mitochondria-related genes by RNA-seq, indicating that mitochondria became active during differentiation. Supporting this notion, the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS) was increased as well. Functionally, we utilized chemicals and genome editing techniques, which could interfere with mitochondrial homeostasis, to determine the role of mitochondria in human endoderm differentiation. Treatment with mitochondrial inhibitors, or genetic depletion of mitochondrial transcription factor A (TFAM), significantly reduced the differentiation efficiency of definitive endoderm. In addition, the defect in endoderm differentiation due to dysfunctional mitochondria could be restored to some extent by the addition of ATP. Moreover, the clearance of excessive ROS due to dysfunctional mitochondria by N-acetylcysteine (NAC) improved the differentiation as well. We further found that ATP and NAC could partially replace the growth factor activin A for definitive endoderm differentiation. Our study illustrates the essential role of mitochondria during human endoderm differentiation through providing ATP and regulating ROS levels, which may provide new insight for metabolic regulation of cell fate determination.
Highlights
Mitochondria are considered highly plastic organelles, which continuously alter their morphology by undergoing dynamic processes to respond to cellular demands [1]
Mitochondria provide the necessary energy for various life activities and play a vital role in development, tumorigenesis, immune regulation, and other biological processes [48,49,50,51]
We investigated the role of mitochondria during the definitive endoderm (DE) differentiation of human Pluripotent stem cells (PSCs)
Summary
Mitochondria are considered highly plastic organelles, which continuously alter their morphology by undergoing dynamic processes to respond to cellular demands [1]. Mitochondria are rare organelles with their own genome, some of which are required for the respiratory activity to supply the cell with metabolic energy [2]. Mitochondria provide reaction sites for many catabolism and anabolism, such as the tricarboxylic acid (TCA) cycle, fatty acid oxidation, and certain phospholipid synthesis [4]. These metabolic processes provide reactive raw materials for energy production and produce some metabolites involved in all aspects of cell activity. A few studies suggested that mitochondrial dysfunction led to impaired energy production and contributed to the regulation of various cell activities [5, 6]. Studies on the role of mitochondria in various biological processes and disease pathogenesis have attracted great attention
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