Introduction: Direct reprogramming of somatic cells into other lineages without going through an intermediate pluripotent state is a promising approach for manipulating cell fate, offering both speed and efficiency in generating specific cell types. We have previously shown that a single transcription factor, NeuroD1, can directly convert mouse microglia into neurons within a week. Furthermore, in vivo reprogramming of microglia in a stroke model mouse significantly improved neurological function. However, neuronal conversion of human microglia, which is critically important for clinical applications, has not yet been achieved. In this study, we attempt to address this gap.

Methods & Results: Firstly, microglia were differentiated from human induced pluripotent stem cells (hiPSCs) and confirmed to have microglial characteristics based on gene expression patterns. While hiPSCs could differentiate into neurons with nearly 100% efficiency upon NeuroD1 expression, hiPSC-derived microglia (hiPSMG) were hardly converted into neurons despite high expression of NeuroD1. Therefore, we hypothesized that, unlike mouse microglia, a reprogramming barrier that inhibits the pioneer function of NeuroD1 is constructed during the differentiation of hiPSCs into human microglia. To explore the human-specific reprogramming barrier, we performed genome-wide CRISPR-Cas9 knockout screening in hiPSMG. As a result, random gene knockout allowed NeuroD1 expression to induce a small but detectable number of neurons from hiPSMG. We are currently investigating sgRNA enrichment in converted neurons to identify candidate barrier genes. We also plan to knock out these candidate genes in hiPSMG and assess neuronal conversion efficiency via NeuroD1 expression. Additionally, we have established in vivo-like experimental platforms focused on human microglia, including brain organoids co-cultured with hiPSMG, and immunodeficient mice transplanted with hiPSMG. We will perform direct neuronal conversion in these in vivo conditions.

Conclusion: We have revealed that human microglia establish a reprogramming barrier against NeuroD1. We are actively working to identify and overcome this reprogramming barrier to enable successful neuronal conversion of human microglia. If successful, our study will provide critical insights into human microglial reprogramming and bring us one step closer to developing novel therapeutic strategies for neurological disorders.