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.

Disclosure

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

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