Tamara Major

Oligodendrocytes are the main myelinating cell of the adult CNS and are vulnerable to injury in diverse disorders, such as spinal cord injury, stroke, trauma, pharmacological and radiation toxicity, as well as neuroinflammation. Human pluripotent stem cells are attractive sources of oligodendrocyte lineage cells and provide a promising treatment strategy for exogenous myelin repair through transplantation. This unit describes a protocol for the step‐wise differentiation of forebrain late oligodendrocyte progenitor cells (OPCs) from human pluripotent stem cells in defined chemical in vitro culture conditions. It involves a stepwise progression of oligodendrocyte progenitors through their known developmental phases, starting with the expression of appropriate transcription factors (Olig2, Nkx2.2), the upregulation of PDGFRA, followed by the appearance of O4‐expressing cells, then O1 expression and finally mature myelin‐binding protein (MBP) expressing cells. Validation of cell fate is performed by extensive transcriptomal profiling, as well in vitro myelination essays with hESCs‐derived neuronal cells. Recapitulating forebrain oligodendrocyte development may generate cells more suitable for transplantation strategies for disorders primarily involving the telencephalon. © 2016 by John Wiley & Sons, Inc.

Over 70% of diffuse intrinsic pediatric gliomas, an aggressive brainstem tumor, harbor heterozygous mutations that create a K27M amino acid substitution (methionine replaces lysine 27) in the tail of histone H3.3. The role of the H3.3K27M mutation in tumorigenesis is not fully understood. Here, we use a human embryonic stem cell system to model this tumor. We show that H3.3K27M expression synergizes with p53 loss and PDGFRA activation in neural progenitor cells derived from human embryonic stem cells, resulting in neoplastic transformation. Genome-wide analyses indicate a resetting of the transformed precursors to a developmentally more primitive stem cell state, with evidence of major modifications of histone marks at several master regulator genes. Drug screening assays identified a compound targeting the protein menin as an inhibitor of tumor cell growth in vitro and in mice.

The derivation of induced human pluripotent stem cells (hiPS) has generated significant enthusiasm particularly for the prospects of cell-based therapy. But there are concerns about the suitability of iPS cells for in vivo applications due in part to the introduction of potentially oncogenic transcription factors via viral vectors. Recently developed lentiviral vectors allow the excision of viral reprogramming factors and the development of transgene-free iPS lines. However it is unclear if reprogramming strategy has an impact on the differentiation potential and the in vivo behavior of hiPS progeny. Here we subject viral factor-free, c-myc-free and conventionally reprogrammed four-factor human iPS lines to a further challenge, by analyzing their differentiation potential along the 3 neural lineages and over extended periods of time in vitro, as well as by interrogating their ability to respond to local environmental cues by grafting into the striatum. We demonstrate similar and efficient differentiation into neurons, astrocytes and oligodendrocytes among all hiPS and human ES line controls. Upon intracranial grafting in the normal rat (Sprague Dawley), precursors derived from all hiPS lines exhibited good survival and response to environmental cues by integrating into the subventricular zone, acquiring phenotypes typical of type A, B or C cells and migrating along the rostral migratory stream into the olfactory bulb. There was no teratoma or other tumor formation 12 weeks after grafting in any of the 26 animals used in the study. Thus neither factor excision nor persistence of c-myc impact the behavior of hiPS lines in vivo.

Glioblastoma multiforme (GBM) is a highly heterogeneous malignant tumor. Recent data suggests the presence of a hierarchical organization within the GBM cell population that involves cancer cells with stem-like behavior, capable of repopulating the tumor and contributing to its resistance to therapy. Tumor stem cells are thought to reside within a vascular niche that provides structural and functional support. However, most GBM studies involve isolated tumor cells grown under various culture conditions. Here, we use a novel three-dimensional organotypic “explant” system of surgical GBM specimens that preserves cytoarchitecture and tumor stroma along with tumor cells. Notch inhibition in explants results in decreased proliferation and self-renewal of tumor cells but is also associated with a decrease in endothelial cells. When endothelial cells are selectively eliminated from the explants via a toxin conjugate, we also observed a decrease in self-renewal of tumor stem cells. These findings support a critical role for tumor endothelial cells in GBM stem cell maintenance, mediated at least in part by Notch signaling. The explant system further highlighted differences in the response to radiation between explants and isolated tumor neurospheres. Combination treatment with Notch blockade and radiation resulted in a substantial decrease in proliferation and in self-renewal in tumor explants while radiation alone was less effective. This data suggests that the Notch pathway plays a critical role in linking angiogenesis and cancer stem cell self-renewal and is thus a potential therapeutic target. Three-dimensional explant systems provide a novel approach for the study of tumor and microenvironment interactions.

ATP-dependent oligomeric proteases are major components of cellular protein quality control systems. To investigate the role of proteolytic processes in the maintenance of mitochondrial functions, we analyzed the dynamic behavior of the mitochondrial proteome of Saccharomyces cerevisiae by two-dimensional (2D) polyacrylamide gel electrophoresis. By a characterization of the influence of temperature on protein turnover in isolated mitochondria, we were able to define four groups of proteins showing a differential susceptibility to proteolysis. The protein Pim1/LON has been shown to be the main protease in the mitochondrial matrix responsible for the removal of damaged or nonnative proteins. To assess the substrate range of Pim1 under in vivo conditions, we performed a quantitative comparison of the 2D protein spot patterns between wild-type and pim1Δ mitochondria. We were able to identify a novel subset of mitochondrial proteins that are putative endogenous substrates of Pim1. Using an in organello degradation assay, we confirmed the Pim1-specific, ATP-dependent proteolysis of the newly identified substrate proteins. We could demonstrate that the functional integrity of the Pim1 substrate proteins, in particular, the presence of intact prosthetic groups, had a major influence on the susceptibility to proteolysis.

The protease Pim1/LON, a member of the AAA+ family of homo-oligomeric ATP-dependent proteases, is responsible for the degradation of soluble proteins in the mitochondrial matrix. To establish the molecular parameters required for the specific recognition and proteolysis of substrate proteins by Pim1, we analyzed thein organellodegradation of imported reporter proteins containing different structural properties. The amino acid composition at the amino-terminal end had no major effect on the proteolysis reaction. However, proteins with an amino-terminal extension of less than 60 amino acids in front of a stably folded reporter domain were completely resistant to proteolysis by Pim1. Substrate proteins with a longer amino-terminal extension showed incomplete proteolysis, resulting in the generation of a defined degradation fragment. We conclude that Pim1-mediated protein degradation is processive and is initiated from an unstructured amino-terminal segment. Resistance to degradation and fragment formation was abolished if the folding state of the reporter domain was destabilized, indicating that Pim1 is not able to unravel folded proteins for proteolysis. We propose that the requirement for an exposed, large, non-native protein segment, in combination with a limited unfolding capability, accounts for the selectivity of the protease Pim1 for damaged or misfolded polypeptides.

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism resulting from the absence or dysfunction of a copper transporting P-type ATPase (ATP7B). Approximately 150 mutations of the ATP7B have been identified to date. In this paper, we report the results of molecular characterization and genotype-phenotype analysis, which we have carried out on 35 patients from Yugoslavia affected by WD. Using single-strand conformational polymorphism (SSCP) followed by direct sequencing, we characterized the molecular defect in 80% of WD chromosomes and found 11 different mutations, three of which are novel. The most common mutations that accounted for the molecular defect in 71.3% of WD chromosomes were H1069Q (48.9%), 2304-2305insC (11.4%), R616Q (5.7%), and A1003T (5.7%). The results produced in this paper indicate that the best strategy for mutation detection in Yugoslavian patients with WD is an SSCP analysis of exons 14, 8, 5, and 13, where most of the defects (73.1%) lie, followed by mutation analysis of the remaining exons in ATP7B in patients in whom the mutation was not detected by the finitial screening. These data can be used to develop straightforward genetic testing in this population or in other countries composed of a genetically mixed population like the United States, where a significant number of immigrants came from Central and Eastern Europe.