To compare the transcriptional profiles lineage-reprogrammed and primary cells, an unsupervised machine-learning approach based on principal component analysis (PCA) was used to select genes that better correspond to different cell states. 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) (GEO: GSE67310); 2) Primary neurons from postnatal mice brains (P7) (GEO: GSE52564); and 3) neural progenitors and primary neurons from embryonic mice brains (GEO: GSE65487). As each gene represents a dimension, a strategy based on a dimensional reduction was applied. Thus, samples were ordered to create a pseudo-temporal map, which places cells towards a cell differentiation state (Fig. 1A).
Interestingly, we observed that the transcriptional profiles of reprogrammed induced neurons and primary neurons fully overlapped (Fig. 1A). More interestingly, a continuum was observed from MEFs to a subpopulation of Ascl1- or BAM-transduced cells after 5 and 22 days, where part of the cells overlap with primary neural progenitors and immature neurons, whereas other cells follow a different path (Fig. 1A). This first analysis suggests that the transcriptional programs enacted during the conversion from MEFs to induced neurons and neural progenitor cells to neurons are similar.
To further confirm that the observed pseudo-temporal map could represent different fates of reprogrammed MEFs, we first analyzed cell-type ontologies on Mouse Gene Atlas using enrichR (Fig. 1B). We could confirm that genes were enriched for MEF, neural cells and muscle cell ontologies (Fig. 1B). Next, pan-neuronal markers (Tubb3, Map2), myocyte markers (Tnnc2, Myo18b) and MEF markers (S100a4, Col1a2) were selected among the 400 genes to analyze the levels of expression in different cell states (Fig. 1C and D). S100a4 and Col1a2 were more expressed in the branch containing MEFs, whereas Tnnc2 and Myo18b were more expressed in the branch comprising most ascl1d5 and a small subset of bamd22 cells. In contrast, Tubb3 and Map2 expression were enriched in the branch comprising few ascl1d5, the majority of bamd22 and primary neural progenitors and neurons. These observations suggest that the node (number one) identified in the pseudo-temporal map pinpoints the divergence between cells following a muscular- or neuronal-cell fate.
Next, we set out to identify genes enriched in the ascl1d5 cells classified in the neuronal branch as compared to the MEF and muscle-cell branches. Thus, gene expression patterns of ascld1d5 cell populations classified in each of those branches were compared. We identified 33 genes differentially expressed (q-value <0.05, likelihood ratio test) in the three different populations of ascl1d5 cells (Fig. 1E). Among these genes, 4 were highly enriched in ascl1d5 cells in the neuronal branch (Fig. 1F). Interestingly, these genes were also enriched in bamd22 cells and primary neural cells (Fig. 1F).
In this work, we show that lineage-reprogrammed MEFs undergo transcriptional changes towards the generation of induced neurons that resembles those observed in the transition from primary cerebral cortex progenitors to early-differentiated neurons. 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, 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. We also show that three different populations of ascl1d5 cells can be distinguished based on their transcriptional profiles. Gene expression patterns of these populations are classified in the unsupervised machine-learning approach in branches containing either undifferentiated MEFs, muscle cells or neurons. Using this classification, we identified 33 genes differentially expressed in ascl1d5 cell populations and four genes specifically enriched in ascl1d5 cells in the neuronal branch. These genes may be interesting candidates or contribute to identify new factors to enhance MEF lineage-reprogramming into iNs.
Collapsin Response Mediator Protein 1 (Crmp1) is part of CRMP family of proteins and is typically associated as mediator of sema3A signaling and axon guidance. Interestingly, some CRMP proteins are diferentially expressed in axon and dendrites of distinct neuronal types. Embryonic Lethal, Abnormal Vision, Drosophila-Like 4 (Elavl4), also known as Hu-Antigen D (HuD) is a RNA-binding protein involved in neuronal maturation, neurite outgrowth and dendritic maintenance. Stathmin 3 (Stmn3) or SCG10-Like Protein (SCLIP) is also related to dendritic formation and neurite outgrowth. Zinc finger, CCHC domain containing 12 (Zcchc12) or Smad-Interacting Zinc Finger Protein 1 (Szn1) is a protein used in BMP, AP-1 and CREB signalling as a co-activator. Possibly, Brn2 and Myt1l may sustain gene expression of those candidate genes longer than Ascl1-only reprogramming which allows MEF cells differentiate into neuron-like cells. Thereby, all candidates are related to neuronal phenotype at some level, which indicate possible proteins to help Ascl1 reprogramming MEFs achieve a neuron-like state.