How signaling pathways control embryonic stem cell (ESC) differentiation is poorly understood. Recent studies suggested mitochondria metabolism may play a role in the regulation of stem cell differentiation. In this study, we found a novel role for mitochondrial Akt1 (mito-Akt) that modulated human ESC (hESC) differentiation lineage. We observed that Akt was activated and translocated into mitochondria in hESC upon growth factor stimulation. To determine the effect of mito-Akt on hESC differentiation, hESC were transduced with His-tagged mitochondrial-targeting dominant negative Akt1/GFP (Ad-mdnAkt) or GFP adenoviral vector (Ad-GFP). The transgene was expressed in >90% cells, and mitochondria-targeting was verified. Ad-mdnAkt altered hESC respiration, as measured with Seahorse extracellular flux analyzer, and mitochondria proton leak was increased. After 21 days of differentiation in vitro, H&E stain showed Ad-mdnAkt transduced cells formed more organized cell clusters than control ones. Bulk RNA was extracted for qRT-PCR with primer array of representative marker genes, and the results suggested that marker genes for mesoderm and endoderm were up-regulated whereas marker genes for ectoderm were down-regulated when mito-Akt was inhibited by Ad-mdnAkt in hESCs. To further dissect how mito-Akt modulates cell lineage, single-cell RNA seq was analyzed with 10X Genomics platform. Among the 24 clusters identified by t-SNE plot, Ad-mdnAkt cells were over-represented in 5 clusters and under-represented in 4 clusters when compared to the controls. Interestingly, hESCs with Ad-mdnAkt favored lineages toward hematological system and lymphoid tissue development instead of pancreatic progenitor cells.

In summary, mito-Akt is a novel modulator of stem cell differentiation, altering mito-Akt signaling during the early phase of hESC differentiation may impact later embryonic development.


H. Lee: None. Y. Chen: None. H.Y. Lin: None. A. Ta: None. P.H. Wang: None. Y. Chen: None. B. Andersen: None.

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at