HOPX regulates the development and lineage progression of human stem cell-derived outer radial glia-like cells.

Outer radial glia (oRG) are abundant in the human fetal cortex but rare in rodents, yet their molecular regulation remains poorly understood. Although the homeodomain-only protein X (HOPX) is a well-established oRG marker, its functional role in human cortical development remains undefined. Using CRISPR-Cas9 gene editing in human pluripotent stem cells, we show that HOPX loss impairs the expansion and lineage progression of oRG-like cells in two-dimensional neural cultures and three-dimensional cortical organoids. HOPX knockout precursors exhibit reduced proliferation, diminished neurogenesis, and premature differentiation toward astrocytic and oligodendrocytic lineages. HOPX-deficient organoids display reduced outer subventricular zone size, decreased neuronal output, and early gliogenesis. Mechanistically, HOPX loss impairs PI3K-mTOR signaling. Transcriptomic analysis identifies HOPX-regulated programs governing gliogenic competence, mTOR, and Wnt signaling. Together, these findings identify HOPX as an important regulator of oRG-like cell maintenance and the neurogenic-gliogenic balance, providing insight into the molecular programs governing human neural progenitor expansion.