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Recently, direct reprogramming between divergent lineages has been achieved by the introduction of regulatory transcription factors. This approach may provide alternative cell resources for drug discovery and regenerative medicine, but applications could be limited by the genetic manipulation involved. Here, we show that mouse fibroblasts can be directly converted into neuronal cells using only a cocktail of small molecules, with a yield of up to textgreater90% being TUJ1-positive after 16 days of induction. After a further maturation stage, these chemically induced neurons (CiNs) possessed neuron-specific expression patterns, generated action potentials, and formed functional synapses. Mechanistically, we found that a BET family bromodomain inhibitor, I-BET151, disrupted the fibroblast-specific program, while the neurogenesis inducer ISX9 was necessary to activate neuron-specific genes. Overall, our findings provide a proof of principle" for chemically induced direct reprogramming of somatic cell fates across germ layers without genetic manipulation�

Targeting native progenitors with small molecule pharmaceuticals that direct cell fate decisions is an attractive approach for regenerative medicine. Here, we show that 3,5-disubstituted isoxazoles (Isx), stem cell-modulator small molecules originally recovered in a P19 embryonal carcinoma cell-based screen, directed cardiac muscle gene expression in vivo in target tissues of adult transgenic reporter mice. Isx also stimulated adult mouse myocardial cell cycle activity. Narrowing our focus onto one target cardiac-resident progenitor population, Isx directed muscle transcriptional programs in vivo in multipotent Notch-activated epicardium-derived cells (NECs), generating Notch-activated adult cardiomyocyte-like precursors. Myocardial infarction (MI) preemptively differentiated NECs toward fibroblast lineages, overriding Isx's cardiogenic influence in this cell population. Isx dysregulated gene expression in vivo in Notch-activated repair fibroblasts, driving distinctive (pro-angiogenesis) gene programs, but failed to mitigate fibrosis or avert ventricular functional decline after MI. In NECs in vitro, Isx directed partial muscle differentiation, which included biosynthesis and assembly of sarcomeric α-actinin premyofibrils, beaded structures pathognomonic of early developing cardiomyocytes. Thus, although Isx small molecules have promising in vivo efficacy at the level of cardiac muscle gene expression in native multipotent progenitors and are first in class in this regard, a greater understanding of the dynamic interplay between fibrosis and cardiogenic small molecule signals will be required to pharmacologically enable regenerative repair of the heart.

Adult neurogenesis occurs throughout life in the mammalian hippocampus and is essential for memory and mood control. There is significant interest in identifying ways to promote neurogenesis and ensure maintenance of these hippocampal functions. Previous work with a synthetic small molecule, isoxazole 9 (Isx-9), highlighted its neuronal-differentiating properties in vitro. However, the ability of Isx-9 to drive neurogenesis in vivo or improve hippocampal function was unknown. Here we show that Isx-9 promotes neurogenesis in vivo, enhancing the proliferation and differentiation of hippocampal subgranular zone (SGZ) neuroblasts, and the dendritic arborization of adult-generated dentate gyrus neurons. Isx-9 also improves hippocampal function, enhancing memory in the Morris water maze. Notably, Isx-9 enhances neurogenesis and memory without detectable increases in cellular or animal activity or vascularization. Molecular exploration of Isx-9-induced regulation of neurogenesis (via FACS and microarray of SGZ stem and progenitor cells) suggested the involvement of the myocyte-enhancer family of proteins (Mef2). Indeed, transgenic-mediated inducible knockout of all brain-enriched Mef2 isoforms (Mef2a/c/d) specifically from neural stem cells and their progeny confirmed Mef2's requirement for Isx-9-induced increase in hippocampal neurogenesis. Thus, Isx-9 enhances hippocampal neurogenesis and memory in vivo, and its effects are reliant on Mef2, revealing a novel cell-intrinsic molecular pathway regulating adult neurogenesis.