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

Abstract

Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states1–3. However, the intermediates through which individual cells progress are largely undefined. Here we used single-cell RNA-seq4–7 at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) 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 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.

Direct lineage reprogramming via pioneer factors; a detour through developmental gene regulatory networks

“…In this case, the hematopoietic stem and progenitor GRNs transiently appear at the population level during the course of conversion . In addition, a recent report demonstrated that Ascl1, a neural pioneer factor, activates a neural progenitor-like state and myogenic program when overexpressed in fibroblasts (Treutlein et al, 2016). Although these engineered cell populations might not represent bona fide embryonic progenitors, it is nonetheless clear that they exist due to the engagement of developmental programs at some level.…”

Transcriptional profile of induced and primary neurons reveals new candidate genes for lineage reprogramming

“…5 days after expression of Ascl1 in MEFs, a subset of cells show enriched expression of pan-neuronal genes and are transcriptionally similar to neural progenitors. Similarly, the transcriptional profile of bamd22 cells, which mostly adopt a iN phenotype [12], is closely related to P7 cerebral cortex neurons. In contrast, lineage-reprogrammed MEFs that express low levels of pan-neuronal genes showed enriched expression of fibroblast genes or muscle-cell genes, indicating that those two populations represent MEFs that failed to undergo lineage-conversion or followed an alternative fate [12].…”

“…Similarly, the transcriptional profile of bamd22 cells, which mostly adopt a iN phenotype [12], is closely related to P7 cerebral cortex neurons. In contrast, lineage-reprogrammed MEFs that express low levels of pan-neuronal genes showed enriched expression of fibroblast genes or muscle-cell genes, indicating that those two populations represent MEFs that failed to undergo lineage-conversion or followed an alternative fate [12]. We also show that three different populations of ascl1d5 cells can be distinguished based on their transcriptional profiles.…”

“…We analyzed three datasets: 1) Single-cell RNAseq of mouse embryonic fibroblast (MEF), MEF 5 days after transduction with Ascl1 (ascl1d5), MEF 22 days after transduction with Ascl1 (ascl1d22) and MEF 22 days after transduction with Brn2, Ascl1 and Myt1l (bamd22) [12] (GEO: GSE67310); 2) Primary neurons from postnatal mice brains (P7) [13] (GEO: GSE52564); and 3) neural progenitors and primary neurons from embryonic mice brains [14] (GEO: GSE65487). As each gene represents a dimension, a strategy based on a dimensional reduction was applied.…”

“…As a consequence, genes weakly regulated upon neurogenic transcription factor expression, which may be pivotal for reprogramming, are likely overlooked. More recently, the first comprehensive transcriptional analysis of neuronal induction was published [12]. This work showed that mouse embryonic fibroblasts (MEF) expressing only Ascl1 mostly failed to go through a complete differentiation into iN, going instead towards a myogenic phenotype.…”