# Molecular characterization of directly reprogrammed neurons from human fibroblasts using single cell RNA sequencing

## TL;DR

This study uses single-cell RNA sequencing to track how human skin cells become neurons, revealing molecular changes and potential ways to improve the process.

## Contribution

The study provides a detailed molecular characterization of human fibroblast-to-neuron reprogramming using scRNA-seq and identifies potential targets to enhance maturation.

## Key findings

- PTBP1 knockdown leads to neurogenic transcription factor upregulation and neuronal morphology in human fibroblasts.
- scRNA-seq reveals distinct immature and mature neuronal populations and divergent transcriptional gradients toward neuronal or myofibroblast-like states.
- Transcription factors distinguishing mature from immature neurons are identified, offering insights into maturation barriers.

## Abstract

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.

The online version contains supplementary material available at 10.1038/s41598-025-34688-8.