Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq

Abstract

The transcriptome changes driving the conversion of fibroblasts to neurons at the single-cell level are reported, revealing that early neuronal reprogramming steps are homogenous, driven by the proneural pioneer factor Ascl1; the expression of myogenic genes then has a dampening effect on efficiency, which needs to be counteracted by the neuronal factors Myt1l and Brn2 for more efficient reprogramming. The paths taken by cells undergoing direct conversion from one lineage to another via the expression of reprogramming factors are still undefined. These authors dissect the transcriptome changes driving the process of direct conversion from fibroblasts to neurons at the single cell level and at multiple time points. They find that surprisingly, the early steps occur in a homogenous fashion, driven by the proneural pioneer factor Ascl1. At later stages of conversion, the emergence of expression of genes characteristic of the myogenic lineage has a dampening effect on efficiency, which needs to be counteracted by the neuronal factors Myt1l and Brn2 for efficient reprogramming. Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states1,2,3. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing4,5,6,7 at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.