Autophagy plays a critical role in maintaining cellular homeostasis and regulating stem cell fate. Although microRNAs (miRNAs) are being increasingly recognized for their ability to modulate autophagy and pluripotency in human pluripotent stem cells (hPSCs), the underlying molecular mechanisms remain incompletely understood. This study defines a role for miR-150 deficiency in biasing ectoderm-mesoderm lineage specification in hPSCs and identifies SOX2 as a key downstream node regulated primarily through altered mRNA stability, with additional involvement of an autophagy-associated pathway. miR-150-deficient hPSCs were generated by CRISPR/Cas9-mediated deletion of the miR-150 locus. A range of molecular and cellular techniques were used to investigate how miR-150 regulates SOX2 and KLHL20 expression, modulates the KLHL20-ULK1 interaction, and influences autophagy-mediated SOX2 degradation. Directed differentiation protocols were used to determine the role of miR-150 in germ layer lineage specification. The loss of miR-150 led to increased SOX2 expression by stabilizing its mRNA, resulting in elevated SOX2 protein levels in hPSCs. Concurrently, miR-150 deficiency increased KLHL20 mRNA stability, disrupted ULK1-mediated autophagy, and inhibited SOX2 degradation. Functional assays demonstrated that miR-150 fine-tunes SOX2 expression through dual regulation of mRNA stability and autophagy via the KLHL20-ULK1 axis, maintaining a balanced commitment to the mesodermal and ectodermal lineages. These findings establish miR-150 as a regulator of SOX2 activity and autophagy in hPSCs. By targeting both SOX2 and the KLHL20-ULK1 axis, miR-150 coordinates SOX2 turnover through complementary mechanisms, ensuring precise mesodermal and ectodermal lineage commitment. This multiregulatory strategy provides new insights into how miRNAs integrate intracellular signaling pathways to direct stem cell fate decisions.
