<p>Direct neuronal reprogramming offers a promising strategy to generate functional neurons from somatic cells without passing through a pluripotent state. However, the cellular heterogeneity and molecular transitions underlying this process remain incompletely understood in human cells. Here, we transduced human dermal fibroblasts with lentiviral short hairpin RNA targeting polypyrimidine tract-binding protein 1 (PTBP1) and performed single-cell RNA sequencing (scRNA-seq) at day 14 post-transduction. PTBP1 knockdown was associated with neurogenic transcription factor upregulation and the emergence of neuronal morphology, with conversion efficiency reaching approximately 20% by week 2. scRNA-seq revealed a progression from fibroblasts toward transcriptionally distinct immature and mature neuronal populations, and pseudotime analysis provided a descriptive ordering of cells along divergent transcriptional gradients that reflect transitions toward either neuronal or myofibroblast-like states. Using an analytic framework designed to ensure statistical robustness in single-sample data, we identified transcription factors that reliably distinguish mature from immature neuronal lineages. These findings provide mechanistic insight into barriers to neuronal maturation and highlight potential molecular targets that may improve the efficiency of direct neuronal reprogramming in human cells.</p>