Here we show that conventional reprogramming towards pluripotency through overexpression of Oct4, Sox2, Klf4 and c-Myc can be shortcut and directed towards cardiogenesis in a fast and efficient manner. With as little as 4 days of transgenic expression of these factors, mouse embryonic fibroblasts (MEFs) can be directly reprogrammed to spontaneously contracting patches of differentiated cardiomyocytes over a period of 11-12 days. Several lines of evidence suggest that a pluripotent intermediate is not involved. Our method represents a unique strategy that allows a transient, plastic developmental state established early in reprogramming to effectively function as a cellular transdifferentiation platform, the use of which could extend beyond cardiogenesis. Our study has potentially wide-ranging implications for induced pluripotent stem cell (iPSC)-factor-based reprogramming and broadens the existing paradigm.
The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineagespecific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. Here we show that transient induction of the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiate fibroblasts into functional neural stem/progenitor cells (NPCs) with appropriate signaling inputs. Compared with induced neurons (or iN cells, which are directly converted from fibroblasts), transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells. Our results provide a unique paradigm for iPSC-factorbased reprogramming by demonstrating that it can be readily modified to serve as a general platform for transdifferentiation.A lthough successful transdifferentiation from one cell type to another by overexpressing lineage-specific genes in vivo (1, 2) and in vitro (3, 4) has been reported, until recently these methods were only effective for fate switching within the major lineages, i.e., ectoderm, mesoderm, and endoderm. However, the generation of iN cells (5) using neural-specific transcription factors has established that interlineage transdifferentiation is also possible in vitro. These transdifferentiation schemes entail overexpression of different sets of lineage-specific transcription factors. A more recent example reported single-factor transdifferentiation of fibroblasts into blood precursors using long-term ectopic expression of OCT4 (6); through extensive binding to the regulatory regions of key hematopoietic genes, OCT4 also appears to be participating in regulating hematopoietic programs acting as a lineage-specific transcription factor in this context. An important aspect of this study is the ability to generate a mitotically active progenitor population that can be further differentiated into a variety of blood cells-a critical feat that has yet to be accomplished in transdifferentiation to neural and endoderm lineages.In an effort to devise a more general transdifferentiation strategy that might give rise to a broad array of unrelated cell typesincluding lineage-specific precursors-we attempted to direct conventional four iPSC-factor-based reprogramming (7, 8) toward alternative outcomes. Specifically, studies indicating that iPSCs are generated in a sequential and stochastic manner (9-11) led us to hypothesize that we might be able to manipulate cells at an early and epigenetically highly unstable state induced by the reprogramming factors. Different conditions could potentially give rise to a multitude ...
The lipid kinase Fab1 governs yeast vacuole homeostasis by generating PtdIns(3,5)P 2 on the vacuolar membrane. Recruitment of effector proteins by the phospholipid ensures precise regulation of vacuole morphology and function. Cells lacking the effector Atg18p have enlarged vacuoles and high PtdIns(3,5)P 2 levels. Although Atg18 colocalizes with Fab1p, it likely does not directly interact with Fab1p, as deletion of either kinase activator-VAC7 or VAC14-is epistatic to atg18⌬: atg18⌬vac7⌬ cells have no detectable PtdIns(3,5)P 2 . Moreover, a 2xAtg18 (tandem fusion) construct localizes to the vacuole membrane in the absence of PtdIns(3,5)P 2 , but requires Vac7p for recruitment. Like the endosomal PtdIns(3)P effector EEA1, Atg18 membrane binding may require a protein component. When the lipid requirement is bypassed by fusing Atg18 to ALP, a vacuolar transmembrane protein, vac14⌬ vacuoles regain normal morphology. Rescue is independent of PtdIns(3,5)P 2 , as mutation of the phospholipid-binding site in Atg18 does not prevent vacuole fission and properly regulates Fab1p activity. Finally, the vacuole-specific type-V myosin adapter Vac17p interacts with Atg18p, perhaps mediating cytoskeletal attachment during retrograde transport. Atg18p is likely a PtdIns(3,5)P 2 "sensor," acting as an effector to remodel membranes as well as regulating its synthesis via feedback that might involve Vac7p. INTRODUCTIONEukaryotic cells have evolved highly sophisticated macromolecular machinery to orchestrate the complex and dynamic process of membrane trafficking. These protein complexes vary widely in structure and function, but are all tightly coordinated in order to catalyze a sequence of specific events with the goal of transferring membrane cargo from one compartment to another. Despite the constant flux of membrane and proteins, organelles maintain distinct identities. To achieve the formation and maintenance of organelle size, shape, and function, trafficking complexes must be properly targeted and subsequently recycled (Munro, 2004;Behnia and Munro, 2005;Jordens et al., 2005). Lipids have been shown to play an increasingly important role in these processes, especially phosphoinositides (PtdInsPs).PtdInsPs are phosphorylated derivatives of phosphatidylinositol, of which there are seven species that can be rapidly interconverted by phosphorylation or dephosphorylation reactions. The occurrence of a specific PtdInsP transiently recruits cognate effector proteins that bind the PtdInsP via short modular motifs such as the FYVE and PH domains (Lemmon, 2003;Balla, 2005;Behnia and Munro, 2005;Di Paolo and De Camilli, 2006;Takenawa and Itoh, 2006). Hence, PtdInsPs are particularly suited to serve as membrane surface tags. Importantly, PtdInsP-binding modules differ greatly in their affinity and specificity for the various PtdInsPs, which when coupled with protein partners, generates a specificity code for each organelle (Lawe et al., 2000;Balla, 2005).PtdIns(3)P and PtdIns(3,5)P 2 , respectively, label early and late endosomal structu...
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