Direct reprogramming of human somatic cells into neuron-like cells involves a transition through a transient intermediate state.

BackgroundDirect conversion of human somatic cells into functional neurons could offer a faster way to generate patient-specific neurons for use in regenerative medicine, disease modelling, and drug development. Although it has been reported that neuronal direct reprogramming bypasses the intermediate pluripotent state, no reports have included time-lapse experiments, potentially overlooking transient intermediate states. Recent studies have shown that the conversion of human mesenchymal stromal cells (hMSCs) into neuron-like cells involves a transition through a transient intermediate state. Therefore, further research is needed to fully understand the process by which human somatic cells can become neurons without cell division. In this study we investigates whether direct neuronal reprogramming of human bone marrow-derived MSC (hBM-MSCs), dental pulp-derived MSC (hDP-MSCs), and adult human dermal fibroblasts (HDFa), involves a transient intermediate state, and sought to further validate the neuronal identity of hMSC-derived induced neurons. MethodsIn this study, we conducted time-lapse experiments to observe the transformation of hBM-MSCs, hDP-MSCs and HDFa, into neurons using a small-molecule-based direct reprogramming protocol. Cellular and ultrastructural changes were further characterized by confocal and electron microscopy. ResultsDirect conversion of hBM-MSCs, hDP-MSCs and HDFa into neuron-like cells occurred rapidly and in absence of cell division. Time-lapse analyses revealed that reprogramming proceeds through a transient intermediate state characterized by distinct morphological changes and dynamic nuclear remodelling. Furthermore, we found that neuron-like cells derived from hBM-MSCs and hDP-MSCs exhibit neuronal polarization, expressed specific neuronal and synaptic markers, formed interconnected cellular networks, and exhibited functional plasticity, providing further evidence that hMSCs can become functional neurons. ConclusionsThis study provides clear evidence that the direct neuronal reprogramming process involves a transition through an intermediate, transient state. Our findings also provide further evidence that hMSCs can become functional neurons. In summary, our work provides new insights into the direct neuronal reprogramming process, which is essential for advancing both developmental biology and regenerative medicine.