Identity crisis: exploring the boundaries of cell type identification in the age of single-cell transcriptomics

The rise of single-cell transcriptomics and comprehensive reference atlases promised a unifying molecular framework to classify cell identity. Yet transcriptomic identities are often interpreted outside the environmental contexts in which they arise. Here, we analyzed primary cortical cultures, which lack native tissue architecture, to compare their transcriptional profiles to multiple <i>in vivo</i> mouse cortical reference datasets. We found that while core molecular signatures for major neuronal subclasses are largely preserved <i>in vitro</i>, the loss of <i>in vivo</i> structure triggers high transcriptional divergence associated with metabolic and physiological state. We also identified clusters that consistently show low confidence in the classification tool. These ambiguous populations express incomplete canonical marker profiles resulting from a lack of structural cues necessary for full maturation. These observations suggest that while transcriptomic reference frameworks capture major aspects of neuronal identity, their interpretation can become less certain when cells are profiled outside their native environment. Our findings highlight the importance of considering environmental context when interpreting transcriptome-based cell type annotations and provide a resource for understanding how neuronal transcriptional programs are reshaped <i>in vitro</i>.