mSEL-1L deficiency affects vasculogenesis and neural stem cell lineage commitment.

mSEL-1L is a highly conserved ER-resident type I protein, involved in the degradation of misfolded peptides through the ubiquitin-proteasome system (UPS), a pathway known to control the plasticity of the vascular smooth muscle cells (VSMC) phenotype and survival. In this article, we demonstrate that mSEL-1L deficiency interferes with the murine embryonic vascular network, showing particular irregularities in the intracranic and intersomitic neurovascular units and in the cerebral capillary microcirculation. During murine embryogenesis, mSEL-1L is expressed in cerebral areas known to harbor progenitor neural cells, while in the adult brain the protein is specifically restricted to the stem cell niches, co-localizing with Sox2 and Nestin. Null mice are characterized by important defects in the development of telenchephalic regions, revealing conspicuous aberration in neural stem cell lineage commitment. Moreover, mSEL-1L depletion in vitro and in vivo appears to affect the harmonic differentiation of the NSCs, by negatively influencing the corticogenesis processes. Overall, the data presented suggests that the drastic phenotypic characteristics exhibited in mSEL-1L null mice can, in part, be explained by the negative influence it plays on Notch1 signaling pathway.

mSEL-1L (Suppressor/enhancer Lin12-like) protein levels influence murine neural stem cell self-renewal and lineage commitment.

Murine SEL-1L (mSEL-1L) is a key component of the endoplasmic reticulum-associated degradation pathway. It is essential during development as revealed by the multi-organ dysfunction and in uterus lethality occurring in homozygous mSEL-1L-deficient mice. Here we show that mSEL-1L is highly expressed in pluripotent embryonic stem cells and multipotent neural stem cells (NSCs) but silenced in all mature neural derivatives (i.e. astrocytes, oligodendrocytes, and neurons) by mmu-miR-183. NSCs derived from homozygous mSEL-1L-deficient embryos (mSEL-1L(-/-) NSCs) fail to proliferate in vitro, show a drastic reduction of the Notch effector HES-5, and reveal a significant down-modulation of the early neural progenitor markers PAX-6 and OLIG-2, when compared with the wild type (mSEL-1L(+/+) NSCs) counterpart. Furthermore, these cells are almost completely deprived of the neural marker Nestin, display a significant decrease of SOX-2 expression, and rapidly undergo premature astrocytic commitment and apoptosis. The data suggest severe self-renewal defects occurring in these cells probably mediated by misregulation of the Notch signaling. The results reported here denote mSEL-1L as a primitive marker with a possible involvement in the regulation of neural progenitor stemness maintenance and lineage determination.

Molecular chaperones as therapeutic targets to counteract proteostasis defects.

The health of cells is preserved by the levels and correct folding states of the proteome, which is generated and maintained by the proteostasis network, an integrated biological system consisting of several cytoprotective and degradative pathways. Indeed, the health conditions of the proteostasis network is a fundamental prerequisite to life as the inability to cope with the mismanagement of protein folding arising from genetic, epigenetic, and micro-environment stress appears to trigger a whole spectrum of unrelated diseases. Here we describe the potential functional role of the proteostasis network in tumor biology and in conformational diseases debating on how the signaling branches of this biological system may be manipulated to develop more efficacious and selective therapeutic strategies. We discuss the dual strategy of these processes in modulating the folding activity of molecular chaperones in order to counteract the antithetic proteostasis deficiencies occurring in cancer and loss/gain of function diseases. Finally, we provide perspectives on how to improve the outcome of these disorders by taking advantage of proteostasis modeling.

The science of stem cell biobanking: investing in the future.

The use of human stem cells in biomedical research projects is increasing steadily and the number of cells that are being derived develops at a remarkable pace. However, stem cells around the world are vastly different in their provenance, programming, and potentials. Furthermore, knowledge on the actual number of cell types, their derivation, availability, and characteristics is rather sparse. Usually, "colleague-supply" avenues constantly furnish cells to laboratories around the world without ensuring their correct identity, characterization, and quality. These parameters are critical if the cells will be eventually used in toxicology studies and drug discovery. Here, we outline some basic principles in establishing a stem cell-specific bank.

Deficiency of suppressor enhancer Lin12 1 like (SEL1L) in mice leads to systemic endoplasmic reticulum stress and embryonic lethality.

Stress in the endoplasmic reticulum (ER) plays an important causal role in the pathogenesis of several chronic diseases such as Alzheimer, Parkinson, and diabetes mellitus. Insight into the genetic determinants responsible for ER homeostasis will greatly facilitate the development of therapeutic strategies for the treatment of these debilitating diseases. Suppressor enhancer Lin12 1 like (SEL1L) is an ER membrane protein and was thought to be involved in the quality control of secreted proteins. Here we show that the mice homozygous mutant for SEL1L were embryonic lethal. Electron microscopy studies revealed a severely dilated ER in the fetal liver of mutant embryos, indicative of alteration in ER homeostasis. Consistent with this, several ER stress responsive genes were significantly up-regulated in the mutant embryos. Mouse embryonic fibroblast cells deficient in SEL1L exhibited activated unfolded protein response at the basal state, impaired ER-associated protein degradation, and reduced protein secretion. Furthermore, markedly increased apoptosis was observed in the forebrain and dorsal root ganglions of mutant embryos. Taken together, our results demonstrate an essential role for SEL1L in protein quality control during mouse embryonic development.

Multilayer and MATR3-dependent regulation of mRNAs maintains pluripotency in human induced pluripotent stem cells.

Matrin3 (MATR3) is a nuclear RNA/DNA-binding protein that plays pleiotropic roles in gene expression regulation by directly stabilizing target RNAs and supporting the activity of transcription factors by modulating chromatin architecture. MATR3 is involved in the differentiation of neural cells, and, here, we elucidate its critical functions in regulating pluripotent circuits in human induced pluripotent stem cells (hiPSCs). MATR3 downregulation affects hiPSCs' differentiation potential by altering key pluripotency regulators' expression levels, including OCT4, NANOG, and LIN28A by pleiotropic mechanisms. MATR3 binds to the OCT4 and YTHDF1 promoters favoring their expression. YTHDF1, in turn, binds the m6A-modified OCT4 mRNA. Furthermore, MATR3 is recruited on ribosomes and controls pluripotency regulating the translation of specific transcripts, including NANOG and LIN28A, by direct binding and favoring their stabilization. These results show that MATR3 orchestrates the pluripotency circuitry by regulating the transcription, translational efficiency, and epitranscriptome of specific transcripts.

Modeling medulloblastoma in vivo and with human cerebellar organoids.

Medulloblastoma (MB) is the most common malignant brain tumor in children and among the subtypes, Group 3 MB has the worst outcome. Here, we perform an in vivo, patient-specific screen leading to the identification of Otx2 and c-MYC as strong Group 3 MB inducers. We validated our findings in human cerebellar organoids where Otx2/c-MYC give rise to MB-like organoids harboring a DNA methylation signature that clusters with human Group 3 tumors. Furthermore, we show that SMARCA4 is able to reduce Otx2/c-MYC tumorigenic activity in vivo and in human cerebellar organoids while SMARCA4 T910M, a mutant form found in human MB patients, inhibits the wild-type protein function. Finally, treatment with Tazemetostat, a EZH2-specific inhibitor, reduces Otx2/c-MYC tumorigenesis in ex vivo culture and human cerebellar organoids. In conclusion, human cerebellar organoids can be efficiently used to understand the role of genes found altered in cancer patients and represent a reliable tool for developing personalized therapies.

Epsins Regulate Mouse Embryonic Stem Cell Exit from Pluripotency and Neural Commitment by Controlling Notch Activation.

Epsins are part of the internalization machinery pivotal to control clathrin-mediated endocytosis. Here, we report that epsin family members are expressed in mouse embryonic stem cells (mESCs) and that epsin1/2 knockdown alters both mESC exits from pluripotency and their differentiation. Furthermore, we show that epsin1/2 knockdown compromises the correct polarization and division of mESC-derived neural progenitors and their conversion into expandable radial glia-like neural stem cells. Finally, we provide evidence that Notch signaling is impaired following epsin1/2 knockdown and that experimental restoration of Notch signaling rescues the epsin-mediated phenotypes. We conclude that epsins contribute to control mESC exit from pluripotency and allow their neural differentiation by appropriate modulation of Notch signaling.

Generation and characterization of a human iPSC line from an ALS patient carrying the Q66K-MATR3 mutation.

Fibroblasts isolated from an Amyotrophic Lateral Sclerosis (ALS)-patient carrying a mutation in Matrin-3 (p.Q66K -MATR3) gene were reprogrammed to the pluripotency stage by using non-integrating episomal plasmids. We generated the Q66K#44DRM induced pluripotent stem cell (iPSC) line that showed regular karyotype, expressed pluripotency-associated markers and were able to properly differentiate into the three germ layers. The heterozygous missense mutation in the MATR3 gene (p.Q66K), which is associated to ALS disease, was present in the generated iPSC line. Resource table.

Establishment of induced pluripotent stem cell (iPSC) line from 55-year old male patient with hemorrhagic Moyamoya disease.

Peripheral blood mononuclear cells (PBMCs) were collected from 55-year old male patient with a confirmed diagnosis of hemorrhagic Moyamoya disease (MMD). PBMCs were reprogrammed using Sendai virus particles delivering the four Yamanaka factors. A footprint-free hiPSC line was characterized by the expression of pluripotency markers and a normal karyotype. These cells were able to give rise to Embryoid Bodies and to a progeny of differentiated cells belonging to the 3 germ layers. This hiPSC line represents a suitable tool for modelling in vitro MMD disease to investigate the cellular mechanisms underlying the occurrence of this pathology.

Establishment of induced pluripotent stem cell (iPSC) line from an 8-year old female patient with ischemic Moyamoya disease.

Peripheral blood mononuclear cells (PBMCs) were collected from an 8-year old female patient affected by ischemic Moyamoya disease (MMD). Patient's PBMCs were reprogrammed using Sendai virus particles delivering the four Yamanaka factors. The footprint free hiPSC line expressed the major pluripotency markers and exhibited a normal karyotype. Cells were competent to give rise to progeny of differentiated cells belonging to the 3 germ layers. This hiPSC line represents a good tool to in vitro model MMD in order to shed light on the cellular and molecular mechanisms responsible for the occurrence of this syndrome.

Human pluripotent stem cells as tools for neurodegenerative and neurodevelopmental disease modeling and drug discovery.

INTRODUCTION: Although intensive efforts have been made, effective treatments for neurodegenerative and neurodevelopmental diseases have not been yet discovered. Possible reasons for this include the lack of appropriate disease models of human neurons and a limited understanding of the etiological and neurobiological mechanisms. Recent advances in pluripotent stem cell (PSC) research have now opened the path to the generation of induced pluripotent stem cells (iPSCs) starting from somatic cells, thus offering an unlimited source of patient-specific disease-relevant neuronal cells. AREAS COVERED: In this review, the authors focus on the use of human PSC-derived cells in modeling neurological disorders and discovering of new drugs and provide their expert perspectives on the field. EXPERT OPINION: The advent of human iPSC-based disease models has fuelled renewed enthusiasm and enormous expectations for insights of disease mechanisms and identification of more disease-relevant and novel molecular targets. Human PSCs offer a unique tool that is being profitably exploited for high-throughput screening (HTS) platforms. This process can lead to the identification and optimization of molecules/drugs and thus move forward new pharmacological therapies for a wide range of neurodegenerative and neurodevelopmental conditions. It is predicted that improvements in the production of mature neuronal subtypes, from patient-specific human-induced pluripotent stem cells and their adaptation to culture, to HTS platforms will allow the increased exploitation of human pluripotent stem cells in drug discovery programs.

Cell and tissue microarray technologies for protein and nucleic acid expression profiling.

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform.

Functional characterization of two secreted SEL1L isoforms capable of exporting unassembled substrate.

SEL1L-A, a transmembrane glycoprotein residing in the endoplasmic reticulum (ER), is a component of the ER-associated degradation (ERAD) pathway. Alternative splicing generates two smaller SEL1L isoforms, -B and -C, that lack the SEL1L-A membrane-spanning region but retain some sel-1-like repeats, known to be involved in multi-protein interactions and signal transduction. In this study the functional characteristics of SEL1L-B and -C were investigated in human cell models. We show that these two isoforms are induced upon ER stress and activation of the unfolded protein response, together with SEL1L-A. Using transient transfection experiments (based on wild-type and mutant SEL1L constructs) combined with several biochemical tests we show that SEL1L-B and, more prominently, SEL1L-C are secreted glycoproteins. Although SEL1L-C is in monomeric form, SEL1L-B is engaged in intramolecular/intermolecular disulfide bonds. Both isoforms localize in secretory and degradative cellular compartments and in areas of cell-cell contact. However, whereas SEL1L-B is mainly associated with membranes, SEL1L-C shows the typical intralumenal localization of soluble proteins and is present in intercellular spaces. Furthermore, because of its peroxisomal domain, SEL1L-C localizes to peroxisomes. Both SEL1L-B and -C are involved in sorting and exporting unassembled Ig-mu(s) but do not affect two other ERAD substrates, the null Hong Kong variant of alpha(1)-antitrypsin, and mutant alpha(1)-AT Z. Overall these findings suggest that SEL1L-B and -C participate to novel molecular pathways that, in parallel with ERAD, contribute to the disposure of misfolded/unfolded or orphan proteins through degradation or secretion.