Enhanced lineage reprogramming towards induced neural stem cells with re-engineered SOX17

Abstract

Forced expression of transcription factors (TFs) can program cellular fate changes. The direct transdifferentiation of induced neural stem cells (iNSCs) from somatic cells with defined TFs has been reported in several studies. iNSCs have great potentials in regenerative medicine and as authentic models for age-associated neurological diseases. Compared to cells derived from induced pluripotent stem cells (iPSCs), iNSCs could be a shortcut for the generation of cell models, avoid oncogenic cell states and circumvent the rejuvenation confounding authentic models of age-associated diseases. However, low efficiency, slow speed, poor reproducibility and an unresolved mechanism of iNSC reprogramming impede their routine applications in research and for clinical studies.

In this study, to enhance and dissect iNSC generation, we aimed to identify artificially evolved and enhanced TFs (eTFs) to boost iNSC reprogramming based on a Brn4, Sox2, Klf4 and c-Myc (BSKM) cocktail. We identified mutant variants of the endoderm factor Sox17 that can effectively produce iNSCs, whereas wild-type Sox2 and Sox17 fail. An engineered eSox17 with three point mutations can drive efficient and rapid generation of iNSCs from fetal, adult and old mouse fibroblasts. These iNSCs maintain long-term self-renewal, express neural stem cell markers and are capable of differentiating into neurons, astrocytes and oligodendrocytes.

By scaling down the number of TFs in the reprogramming cocktail, we found exogenous Brn4 and c-Myc factors are dispensable. eSox17 and Klf4 are necessary and sufficient to generate iNSCs. This two-factor cocktail drives direct reprogramming to neural lineage without passing a pluripotent state, demonstrated by the lack of pluripotency gene activation using an Oct4-GFP reporter and Nanog-CreER lineage tracing system. In addition, metabolic assays revealed that Klf4 induces glycolysis essential for iNSC reprogramming.

To uncover the molecular mechanism underlying the unique ability of eSox17 to generate iNSCs, we investigated transcriptional dynamic and chromatin accessibility during neural reprogramming. Global gene expression and chromatin opening are similar at the early stage of reprogramming with different Sox factors. Compared with Sox2 and Sox17, eSox17 differentially targets genomic loci with canonical Sox:Oct DNA elements and activates a small subset of genes to mediate cell fate change during neural reprogramming. Importantly, exogenous TF silencing is required to mature reprogramming cells into iNSCs at the late stage.

In summary, this study shows eSox17 enables robust reprogramming of somatic cells into iNSCs. The translation of these findings to human iNSC generation could lead to readily available, authentic and personalized cell models for neurodegenerative diseases that bypass the shortcomings associated with iPSC-based strategies. The application of eTFs to generate desired cell types could provide promising cell sources for research and regenerative medicine.