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Aligning single-cell developmental and reprogramming trajectories identifies molecular determinants of reprogramming outcome

ABSTRACT: Cellular reprogramming through manipulation of defined factors holds great promise for large-scale production of cell types needed for use in therapy, as well as for expanding our understanding of the general principles of gene regulation. MYOD-mediated myogenic reprogramming, which converts many cell types into contractile myotubes, remains one of the best characterized model system for direct conversion by defined factors. However, why MYOD can efficiently convert some cell types into myotubes but not others remains poorly understood. Here, we analyze MYOD-mediated reprogramming of human fibroblasts at pseudotemporal resolution using single-cell RNA-Seq. Successfully reprogrammed cells navigate a trajectory with two branches that correspond to two barriers to reprogramming, with cells that select incorrect branches terminating at aberrant or incomplete reprogramming outcomes. Differential analysis of the major branch points alongside alignment of the successful reprogramming path to a primary myoblast trajectory revealed Insulin and BMP signaling as crucial molecular determinants of an individual cell’s reprogramming outcome, that when appropriately modulated, increased efficiency more than five-fold. Our single-cell analysis reveals that MYOD is sufficient to reprogram cells only when the extracellular milieu is favorable, supporting MYOD with upstream signaling pathways that drive normal myogenesis in development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE105211 | GEO | 2018/09/26

REPOSITORIES: GEO

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Project description:Synthetic transcription factors can be applied to many areas of biotechnology, medicine, and basic research.Â Currently, the most common method for engineering synthetic transcription factors has been based on programmable DNA-binding domains of zinc finger proteins, Transcription Activator-Like Effectors (TALEs), and most recently the CRISPR/Cas9 system. These transcription factor platforms consist of the DNA-binding domain fused to potent transcriptional activation domains, most commonly the tetramer of the minimal transactivation domain of the VP16 protein from herpes simplex virus, referred to as VP64. Although many applications are well-suited for the targeted activation of a single gene, genetic reprogramming requires the coordinated regulation of many nodes of natural gene networks as is typically performed by naturally occurring reprogramming factors. Thus we sought to combine principles from each of these approaches by attaching potent transcriptional activation domains to a natural reprogramming factor to increase the efficiency and/or rate of cell fate conversion. In this study, we evaluated the effects of fusing potent activation domains to the transcription factor MyoD, the master regulator of the skeletal myoblast lineage. In certain non-myogenic lineages, MyoD overexpression causes upregulation of the myogenic gene network and conversion to a myoblast phenotype including cell fusion into multinucleated myotubes. Compared to wild-type MyoD, the VP64-MyoD fusion protein induced greater overall reprogramming of global gene expression. This simple approach for increasing the potency of natural reprogramming factors circumvents the need for screening engineered proteins and leads to a more robust cellular reprogramming compared to treatment with the wild type transcription factor. Human dermal fibroblasts were transduced with a single tet inducible lentivirus that expresses either WT-MyoD or VP64-MyoD in response to treatment with doxycycline. Untreated human dermal fibroblast served as the negative control. Gene expression was measured using mRNA-seq, and differential expression was calculated using DESeq. All experiments were performed in biological duplicates.

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