Deconstructing Stepwise Fate Conversion of Human Fibroblasts to Neurons by MicroRNAs: Subseries 1 [Figure 1]

Cell-fate conversion generally presumes the activity of reprogramming effectors to both introduce fate programs of the target cell type and erase the identity of starting cell population. Here, we reveal novel insights into the activity of microRNAs, miR-9/9* and miR-124 (miR-9/9*-124) as reprogramming agents that orchestrate direct conversion of human fibroblasts by first eradicating fibroblast identity and promoting uniform transition to a neuronal state in sequence. Among the direct target genes of miR-9/9*-124, we identify KLF-family transcription factors whose repression is critical for erasing fibroblast fate. The subsequent upregulation of a small nuclear RNA, RN7SK induces accessibilities of chromatin regions and neuronal gene activation, pushing the reprogramming cells to a neuronal state. Our study defines deterministic components in the reprogramming cascade by microRNAs. Overall design: De-identified healthy human adult dermal fibroblasts were transduced with lentivirus to express the brain enriched microRNAs, miR-9/9* and miR-124 and motor neuron-enriched transcription factors ISL1 and LHX3 to directly convert fibroblasts into moto-miNs as previously established (Yoo et al., Nature 2011; Richner et al., Nat. Prot. 2015; Abernathy, Kim, and McCoy et al., 2017). Single cells were collected for RNA-seq and prepared for analysis as described in the Protocols.