No abstract

Intestinal organoids are multicellular crypt-like structures that can be derived from adult intestinal stem cells (ISCs), embryonic stem cells (ESCs) or induced pluripotent stem cells (IPSCs). Here we show that intestinal organoids generated from mouse ESCs were enriched in ISCs and early progenitors. Treatment of these organoids with a γ-secretase inhibitor increased Math1 and decreased Hes1 expression, indicating Notch signaling regulates ISC differentiation in these organoids. Lgr5 and Tert positive ISCs constituted approximately 10% and 20% of the organoids. As found in native tissue, Lgr5 and Tert expressing cells resolved into two discreet populations, which were stable over time. Intestinal organoids derived from cancer-prone Apc(Min/+) mice showed similar numbers of ISCs, but had reduced Math1 expression, indicating a suppressed secretory cell differentiation potential (as found in intestinal tissue). Apc(Min/+) organoids were used to screen epigenetically active compounds for those that increased Math1 expression and organoid differentiation (including HDAC inhibitors, Sirtuin (SIRT) modulators and methyltransferase inhibitors). Broad-spectrum HDAC inhibitors increased both Math1 and Muc2 expression, indicating an ability to promote the suppressed secretory cell differentiation pathway. Other epigenetic compounds had a diverse impact on cell differentiation, with a strong negative correlation between those that activated the secretory marker Muc2 and those that activated the absorptive cell marker Fabp2. These data show that ESC-derived intestinal organoids can be derived in large numbers, contain distinct ISC types and can be used to screen for agents that promote cell differentiation through different lineage pathways.

Despite years of research, the reprogramming of human somatic cells to pluripotency remains a slow, inefficient process, and a detailed mechanistic understanding of reprogramming remains elusive. Current models suggest reprogramming to pluripotency occurs in two-phases: a prolonged stochastic phase followed by a rapid deterministic phase. In this paradigm, the early stochastic phase is marked by the random and gradual expression of pluripotency genes and is thought to be a major rate-limiting step in the successful generation of induced Pluripotent Stem Cells (iPSCs). Recent evidence suggests that the epigenetic landscape of the somatic cell is gradually reset during a period known as the stochastic phase, but it is known neither how this occurs nor what rate-limiting steps control progress through the stochastic phase. A precise understanding of gene expression dynamics in the stochastic phase is required in order to answer these questions. Moreover, a precise model of this complex process will enable the measurement and mechanistic dissection of treatments that enhance the rate or efficiency of reprogramming to pluripotency. Here we use single-cell transcript profiling, FACS and mathematical modeling to show that the stochastic phase is an ordered probabilistic process with independent gene-specific dynamics. We also show that partially reprogrammed cells infected with OSKM follow two trajectories: a productive trajectory toward increasingly ESC-like expression profiles or an alternative trajectory leading away from both the fibroblast and ESC state. These two pathways are distinguished by the coordinated expression of a small group of chromatin modifiers in the productive trajectory, supporting the notion that chromatin remodeling is essential for successful reprogramming. These are the first results to show that the stochastic phase of reprogramming in human fibroblasts is an ordered, probabilistic process with gene-specific dynamics and to provide a precise mathematical framework describing the dynamics of pluripotency gene expression during reprogramming by OSKM.

Significant migration cues are required to guide and contain newly generated rodent subventricular zone (SVZ) neuroblasts as they transit along the lateral ventricles and then through the anterior forebrain to their ultimate site of differentiation in the olfactory bulbs (OBs). These cues enforce strict neuroblast spatial boundaries within the dense astroglial meshwork of the SVZ and rostral migratory stream (RMS), yet are permissive to large-scale neuroblast migration. Therefore, the molecular mechanisms that define these cues and control dynamic interactions between migratory neuroblasts and surrounding astrocytes are of particular interest. We found that deletion of EphA4 and specifically ablation of EphA4 kinase activity resulted in misaligned neuroblasts and disorganized astrocytes in the RMS/SVZ, linking EphA4 forward signaling to SVZ and RMS spatial organization, orientation, and regulation. In addition, within a 3 week period, there was a significant reduction in the number of neuroblasts that reached the OB and integrated into the periglomerular layer, revealing a crucial role for EphA4 in facilitating efficient neuroblast migration to the OB. Single-cell analysis revealed that EPHA4 and its EFN binding partners are expressed by subpopulations of neuroblasts and astrocytes within the SVZ/RMS/OB system resulting in a cell-specific mosaic, suggesting complex EphA4 signaling involving both homotypic and heterotypic cell-cell interactions. Together, our studies reveal a novel molecular mechanism involving EphA4 signaling that functions in stem cell niche organization and ultimately neuroblast migration in the anterior forebrain.

It has long been hypothesized that a defect in the repair of damaged DNA is central to the etiology of Fanconi anemia (FA). Indeed, an increased sensitivity of FA patient-derived cells to the lethal effects of various forms of DNA damaging agents was described over three decades ago [1–3]. Furthermore, the cytological hallmark of FA, the DNA crosslink-induced radial chromosome formation, exemplifies an innate impairment in the repair of these particularly cytotoxic DNA lesions [4]. Precisely defining the collective role of the FA proteins in DNA repair, however, continues to be one of the most enigmatic and challenging questions in the FA field. The first six identified FA proteins (A, C, E, F, G, and D2) harbored no recognizable enzymatic features, precluding association with a specific metabolic process. Consequently, our knowledge of the role of the FA proteins in the DNA damage response has been gleaned primarily through biochemical association studies with non-FA proteins. Here, we provide a chronological discourse of the major FA protein interaction network discoveries, with particular emphasis on the DNA damage response, that have defined our current understanding of the molecular basis of FA.