UC Irvine

The ability of self-renewal and the unlimited potential to differentiate into three germ layers make pluripotent stem cells a key player in regenerative medicine and disease modeling. Recent studies have shown that several signaling pathways and the bioenergetic function of mitochondria play critical roles in the regulation of stem cell differentiation and somatic cell reprogramming. Phosphatidylinositol-3 kinase/Akt signaling (PI3K/Akt) has been previously reported to be critical for maintaining the pluripotency of embryonic stem cells (ESC) via interaction with other signaling pathways, such as LIF/JAK/STAT. It has been shown that Akt translocates into mitochondria after growth factor or cytokine stimulation in differentiated cells. Herein, we demonstrated the translocation of Akt into mitochondria also occurs in human embryonic stem cells (hESCs) after serum stimulation. Intriguingly, our previous data showed that activation of mitochondrial Akt signaling enhanced somatic cell reprogramming. However, the effect of mitochondrial Akt signaling on stem cell differentiation has not yet been investigated.

Testing the effect of temporarily inhibiting mitochondrial Akt1 signaling in hESCs, I transduced cells with the His-tagged mitochondrial-targeting dominant negative Akt1/GFP adenoviral vector (Ad-mdnAkt1/GFP) or the GFP adenoviral vector (Ad-GFP) and differentiated spontaneously in vitro in differentiation medium. Evaluating transduction rate by fluorescence microscopy and flow cytometry and was determined to be over 60% in both groups. Immunoblotting was used to verify mutant Akt1 expression, and respiratory function of cells was evaluated by an extracellular flux analyzer. Spontaneous differentiation in vitro was characterized with H&E staining and showed that Ad-mdnAkt1/GFP treated cells formed more organized colonies than Ad-GFP treated cells. In line with this, bulk RNA analyzed by human cell lineage identification qPCR array showed that marker genes of mesoderm and endoderm were up-regulated and marker genes of ectoderm were down-regulated in Ad-mdnAkt1/GFP treated cells. Further Identification of unique development by single-cell RNA sequencing revealed inhibition of hematological and immune cells differentiation and promotion of lung and kidney development, middle ear morphogenesis and smooth muscle cell differentiation in Ad-mdnAkt1/GFP treated cells.