Abstract: Translational research with non-human primates (NHPs) is widely accepted in central nervous system disease areas because of their close anatomical and physiological similarities to humans, allowing for accurate disease modeling and evaluation of therapeutic candidates. We previously established a chronic stroke model in cynomolgus monkeys and demonstrated the therapeutic effect of AS9102246-00 (AS), a lipid nanoparticle that delivers a transcription factor-encoding mRNA into astrocytes to induce neurogenesis, on motor dysfunction. While in vitro assays under controlled conditions are desirable to evaluate whether the efficacy observed in NHPs can be extrapolated to humans, the application of NHP in vitro models has been limited. Here, we report the generation of astrocytes derived from cynomolgus monkey induced pluripotent stem cells (monkey iPSCs) and in vitro efficacy evaluations of AS using these cells. Monkey iPSCs purchased from the Japanese Collection of Research Bioresources were differentiated into neural progenitor cells (NPCs) using dual SMAD inhibition. The NPCs were cultured in Astrocyte Medium for approximately 10 weeks with regular passaging to generate astrocytes. The majority of the cell population co-expressed GFAP and S100β and exhibited a star-shaped morphology, indicating typical astrocyte characteristics. Additionally, few doublecortin (DCX)-positive cells were observed, suggesting minimal neuronal cell contamination. In efficacy evaluations of AS, we treated iPSC-derived astrocytes and assessed induced-neurogenic effects 7 days post-treatment. The number of DCX-positive cells increased with rising concentrations of AS compared to the control on the same day. These DCX-positive cells exhibited a round cell body and neurite-like structures with very little GFAP expression, indicating neuronal characteristics. Furthermore, we successfully detected the efficacy of AS in human cells, suggesting its neurogenic potential may extend to humans as well. In conclusion, we conducted in vitro translational research using monkey iPSC-derived astrocytes. Our approach utilizing iPSCs has the potential to facilitate similar studies in other cell types, improving estimation of clinical responses in human and reducing the number of in vivo studies in animals.
