Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors

TLDR

Human embryonic stem cells promise regenerative medicine, but current differentiation protocols struggle to capture and expand stable primitive neural precursors with broad differentiation potential and stage‑specific propensity. Synergistic inhibition of GSK3, TGF‑β, and Notch pathways by small molecules rapidly converts hESCs into homogeneous primitive neuroepithelium that self‑renew, retain high neurogenic potential, respond to patterning cues, and integrate in vivo, enabling uniform capture and maintenance of primitive neural stem cells.

Abstract

Human embryonic stem cells (hESCs) hold enormous promise for regenerative medicine. Typically, hESC-based applications would require their in vitro differentiation into a desirable homogenous cell population. A major challenge of the current hESC differentiation paradigm is the inability to effectively capture and, in the long-term, stably expand primitive lineage-specific stem/precursor cells that retain broad differentiation potential and, more importantly, developmental stage-specific differentiation propensity. Here, we report synergistic inhibition of glycogen synthase kinase 3 (GSK3), transforming growth factor β (TGF-β), and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within 1 wk under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor, GSK3 inhibitor (CHIR99021), and TGF-β receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. Our work uniformly captures and maintains primitive neural stem cells from hESCs.