Small molecule-mediated rapid generation of functional human astrocytes: unveiling AKT1-STAT1/3 signaling in...

Astrocytes are essential for maintaining brain homeostasis, as they support neurons, regulate synaptic activity, and mediate immune responses within the central nervous system (CNS). Their role in the pathophysiology of various neurological disorders, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis, is increasingly recognized. Thus, differentiation of astrocytes from human induced pluripotent stem cells (hiPSCs) acts as an important tool for studying disease mechanisms and advancing therapeutic development strategies. However, the prolonged time of up to six months required to generate fully mature astrocytes limits their utility, with most protocols yielding only fetal-like astrocytes or relying on artificial transcription factor overexpression. To address this challenge, we developed a small-molecule-based method using PD0325901 (PD), which enables the rapid generation of mature human astrocytes from gliogenic neural stem cells (NSCs) within a short time of 2-3 weeks, without the need for genetic modification. We found that inhibition of MEK1/2 signaling in NSCs via PD resulted in decreased proliferation, upregulation of astrocytic markers, and acquisition of functionally mature astrocytes. Mechanistically, this differentiation process involved AKT1-dependent phosphorylation and activation of STAT1/3 that is the classical pathway for astrocyte differentiation, along with the nuclear loss of the astrocytic transcriptional repressor OLIG2. Overall, our findings present a novel approach for accelerating astrocyte maturation using a small molecule and reveal a key role for AKT1-STAT1/3 signaling in astrocyte development. By significantly shortening the time required to generate mature human astrocytes, this rapid astrocyte differentiation protocol enables more efficient modeling of neurodegenerative diseases and drug screening efforts.