SUMMARY Differentiation of astrocytes from human stem cells has significant potential for analyzing their role in normal brain function and disease, but existing protocols generate only immature astrocytes. Using early neuralization, we generated spinal cord astrocytes from mouse or human embryonic (ESCs) or induced pluripotent (hiPSCs) stem cells with high efficiency. Remarkably, short exposure to FGF1 or FGF2 was sufficient to direct these astrocytes selectively toward a mature quiescent phenotype, as judged both by marker expression and functional analysis. In contrast, TNFα and IL-1β but not FGFs, induced multiple elements of a reactive phenotype but did not affect maturation. These phenotypically defined, scalable populations of spinal cord astrocytes will be important both for studying normal astrocyte function and for modeling human pathological processes in vitro.

Membrane fusion between vesicles and target membranes involves the zippering of a four-helix bundle generated by constituent helices derived from t- and v-SNAREs found on the target and vesicular membranes. In neurons the protein complexin clamps otherwise spontaneous fusion by SNARE proteins, allowing neurotransmitters and other mediators to be secreted when and where they are needed as this clamp is released. The membrane-proximal accessory helix of complexin is necessary for clamping, but its mechanism of action is unknown. Here, we present experiments using a reconstituted fusion system that suggest a simple model in which the complexin accessory helix forms an alternative four-helix bundle with the t-SNARE near the membrane, preventing the v-SNARE from completing its zippering.

Pathogens use diverse molecular machines to penetrate host cells and manipulate intracellular vesicular trafficking. Viruses employ glycoproteins, functionally and structurally similar to the SNARE proteins, to induce eukaryotic membrane fusion. Intracellular pathogens, on the other hand, need to block fusion of their infectious phagosomes with various endocytic compartments to escape from the degradative pathway. The molecular details concerning the mechanisms underlying this process are lacking. Using both an in vitro liposome fusion assay and a cellular assay, we showed that SNARE-like bacterial proteins block membrane fusion in eukaryotic cells by directly inhibiting SNARE-mediated membrane fusion. More specifically, we showed that IncA and IcmG/DotF, two SNARE-like proteins respectively expressed by Chlamydia and Legionella, inhibit the endocytic SNARE machinery. Furthermore, we identified that the SNARE-like motif present in these bacterial proteins encodes the inhibitory function. This finding suggests that SNARE-like motifs are capable of specifically manipulating membrane fusion in a wide variety of biological environments. Ultimately, this motif may have been selected during evolution because it is an efficient structural motif for modifying eukaryotic membrane fusion and thus contribute to pathogen survival.

In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca 2؉ ) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.

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Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC50 1–2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.

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In most genome-wide linkage studies, implication of a causative disease gene often requires years of expanding the study to more families and finer mapping of the initially described region. Even after such efforts, unobtainable sample sizes can be required to make statistically meaningful conclusions about a single gene. Here we demonstrate that by adding a layer of functional biology to statistical genetic results, this process can be accelerated. The diabetes susceptibility locus (chromosome 18p11) was systematically dissected by using a cell-based secretion assay and RNA interference, and we identified laminin ␣1 to have a role in pancreatic ␤ cell secretion. The screen was extended to identify laminin receptor 1 as a functional partner in regards to ␤ cell function. Our approach can potentially be widely used in the setting of high-throughput cellular screening of other loci to identify candidate genes.diabetes ͉ functional genomics ͉ insulin ͉ laminin ͉ RNA interference