Neural Progenitor Cell Line Research Articles

AG424 and AG427 antibodies recognize the human TrkB protein by western blot

The recombinant antibodies AG424 and AG427 detect the human TrkB protein in the human neural progenitor cell line ReNcell® VM and in human brain by western blot.
 
 

Aberrant mitochondrial function in patient-derived neural cells from CDKL5 deficiency disorder and Rett syndrome.

The X-linked neurodevelopmental diseases CDKL5 deficiency disorder (CDD) and Rett syndrome (RTT) are associated with intellectual disability, infantile spasms and seizures. Although mitochondrial dysfunction has been suggested in RTT, less is understood about mitochondrial function in CDD. A comparison of bioenergetics and mitochondrial function between isogenic wild-type and mutant neural progenitor cell (NPC) lines revealed increased oxygen consumption in CDD mutant lines, which is associated with altered mitochondrial function and structure. Transcriptomic analysis revealed differential expression of genes related to mitochondrial and REDOX function in NPCs expressing the mutant CDKL5. Furthermore, a similar increase in oxygen consumption specific to RTT patient-derived isogenic mutant NPCs was observed, though the pattern of mitochondrial functional alterations was distinct from CDKL5 mutant-expressing NPCs. We propose that aberrant neural bioenergetics is a common feature between CDD and RTT disorders. The observed changes in oxidative stress and mitochondrial function may facilitate the development of therapeutic agents for CDD and related disorders.

Light-Emitting Diode Photobiomodulation After Cerebral Ischemia.

Photobiomodulation (PBM) therapy is a promising therapeutic approach for several pathologies, including stroke. The biological effects of PBM for the treatment of cerebral ischemia have previously been explored as a neuroprotective strategy using different light sources, wavelengths, and incident light powers. However, the capability of PBM as a novel alternative therapy to stimulate the recovery of the injured neuronal tissue after ischemic stroke has been poorly explored. The aim of this study was to investigate the low-level light irradiation therapy by using Light Emitting Diodes (LEDs) as potential therapeutic strategy for stroke. The LED photobiomodulation (continuous wave, 830 nm, 0.2–0.6 J/cm2) was firstly evaluated at different energy densities in C17.2 immortalized mouse neural progenitor cell lines, in order to observe if this treatment had any effect on cells, in terms of proliferation and viability. Then, the PBM-LED effect (continuous wave, 830 nm, 0.28 J/cm2 at brain cortex) on long-term recovery (12 weeks) was analyzed in ischemic animal model by means lesion reduction, behavioral deficits, and functional magnetic resonance imaging (fMRI). Analysis of cellular proliferation after PBM was significantly increased (1 mW) in all different exposure times used; however, this effect could not be replicated in vivo experimental conditions, as PBM did not show an infarct reduction or functional recovery. Despite the promising therapeutic effect described for PBM, further preclinical studies are necessary to optimize the therapeutic window of this novel therapy, in terms of the mechanism associated to neurorecovery and to reduce the risk of failure in futures clinical trials.

Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting.

Genetic and epigenetic variability between iPSC-derived neural progenitor cells (NPCs) combined with differences in investigator technique and selection protocols contributes to variability between NPC lines, which subsequently impacts the quality of differentiated neuronal cultures. We therefore sought to develop an efficient method to reduce this variability in order to improve the purity of NPC and neuronal cultures. Here, we describe a magnetic activated cell sorting (MACS) method for enriching NPC cultures for CD271-/CD133+ cells at both early (<2–3) and late (>10) passage. MACS results in a similar sorting efficiency to fluorescence activated cell sorting (FACS), while achieving an increased yield of live cells and reduced cellular stress. Furthermore, neurons derived from MACS NPCs showed greater homogeneity between cell lines compared to those derived from unsorted NPCs. We conclude that MACS is a cheap technique for incorporation into standard NPC differentiation and maintenance protocols in order to improve culture homogeneity and consistency.

Abstract P1-19-05: Establishment of a human neural progenitor cell microenvironment model to investigate signalling events in triple negative breast cancer brain metastases in a high-throughput setting

Abstract Background. Breast cancer brain metastases (BCBM) represents a terminal diagnosis accompanied by neurological decline and diminished quality of life. Average survival is less than a year following detection of neural lesions, due in part to an absence of targeted therapeutics. BCBM affects 12-17% of all breast cancer patients and over 25% of triple negative breast cancer (TNBC) patients. In the search for improved therapies, there is a focus increasingly on the role of the unique microenvironment of the brain in tumour progression. Few studies have investigated the establishment of an in vitro breast cancer brain microenvironment model that could enable the high-throughput identification and modulation of potential targets. In order to address this issue, a novel co-culture model was developed utilising the human neural progenitor cell (NPC) line ReNcell VM and MDA-MB-468 TNBC cells. Methods. ReNcell VM were differentiated as monolayers or neurospheres for four or ten days and stained for the glial marker glial fibrillary acidic protein (GFAP) and the neuronal marker β3-tubulin. Cultures were imaged via automated epi-fluorescence microscopy (ImageXpress Micro, Molecular Devices) and proportions of cell types were calculated with MetaXpress image analysis software. Differentiation characteristics of neurospheres and monolayers were compared. Functionality of neural cultures was determined using calcium assays with the muscarinic agonist carbachol as a validation tool. MDA-MB-468 cells loaded with a Cell Tracker Green dye were assessed for viability on the neural matrix via bright field and epi-fluorescence microscopy. Results. ReNcell VM cultured and differentiated for four days as neurospheres displayed a higher expression of β3-tubulin and GFAP than those cultured as monolayers (β3-tubulin 10.4% vs 7.7%, GFAP 53.6% vs 48.8%, respectively). Expression of β3 tubulin was higher after four days of differentiation than after ten days for neurosphere cultures (10.4% vs 4.4%, respectively). Carbachol elicited oscillatory calcium responses from ReNcell VM differentiated as neurospheres or monolayers, indicating functionality of both populations. MDA-MB-468 and ReNcell VM stained after four days of co-culture displayed successful integration of neoplastic cells on the neural matrix. Conclusions. Human NPCs provide a reproducible model of prominent cell types within the brain milieu and are suited to high-throughput automated epi-fluorescence microscopy applications. Co-culture models of TNBC and differentiated ReNcell VM represent a powerful platform for mechanistic studies and drug screening. Citation Format: Chalmers SB, Dalley AJ, Kalita-de Croft P, Saunus J, Bassett JJ, Sadras F, Roberts-Thomson SJ, Monteith GR. Establishment of a human neural progenitor cell microenvironment model to investigate signalling events in triple negative breast cancer brain metastases in a high-throughput setting [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-19-05.

SU14 - TRANSCRIPTOME ANALYSIS OF WNT7A-MEDIATED GENE EXPRESSION IN HUMAN NEURAL PROGENITOR CELLS DERIVED FROM CONTROL AND CHD8 HAPLOINSUFFICIENT INDUCED PLURIPOTENT STEM CELLS

Background CHD8 (Chromodomain Helicase DNA Binding Protein 8) codes for a member of the CHD family of chromatin-remodeling factors and is one of the most commonly mutated genes in Autism Spectrum Disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in Schizophrenia (SZ) and intellectual disabilities. We previously reported RNA-seq analyses carried out on Neural Progenitor Cells (NPCs), monolayer neurons and cerebral organoids derived from control induced Pluripotent Stem (iPS) cells that were made haploinsufficient for CHD8 (CHD8+/-) using CRISPR-Cas9 gene editing. Several hundred Differentially Expressed Genes (DEGs) were found in comparisons between the CHD8+/- and isogenic controls (CHD8+/+), including a significant number of ASD and SZ candidate genes, such as the SZ and Bipolar Disorder (BD) candidate gene TCF4. Pathway analysis of DEGs showed enrichment for genes involved in Wnt-signaling. A number of Wnt receptors (members of the FZD family) and Wnt ligands were differentially expressed, such as Wnt7A and its receptor FZD7, which were down-regulated in CHD8+/- NPCs. Methods We tested the hypothesis that Wnt7A-mediated gene expression is deficient in CHD8+/- NPCs by carrying out RNA-seq on two CHD8+/- lines and two controls, including the isogenic control sample. The NPCs were treated with vehicle (control) or Wnt7A (1 microgram/ml) for 24 hours. Biological duplicates were prepared for each sample. RNA was extracted and converted to cDNA for paired-end sequencing. The RNA-seq reads were aligned to the human genes annotated in the GENCODE database (v18). Gene expression levels were determined as transcripts per million (TPM). Of the 12,898 expressed genes, with an average TPM >1 in either controls or CHD8+/− samples, DESeq. 2 was used to identify differentially expressed genes (DEGs) at false discovery rate [FDR] Results We obtained 892 DEGs in the isogenic control NPCs, from a comparison of the Wnt7A treated samples and controls, but only 410 among the CHD8+/- samples, suggesting a potential reduction in Wnt7A signaling in the CHD8+/- samples. Among the DEGs affected by Wnt7A in the isogenic controls, but not in CHD8+/- samples was TCF4. Pathway analysis showed that DEGs from the isogenic controls were enriched for Wnt-beta catenin signaling pathway and acute phase response signaling. In CHD8+/- NPCs, there was less enrichment of both. RNA-seq findings were confirmed for selected genes by quantitative real time PCR. Discussion These preliminary findings suggest that CHD8 haploinsufficiency can attenuate Wnt7A signaling. Wnt/β-catenin signaling plays a key role in brain development and in post-mitotic brain functions. Thus, the attenuation of Wnt7A-inducible genes in CHD8 haploinsufficient NPCs could be mediating some of the effects CHD8 loss-of-function mutations have on brain structures and patient behaviors. Understanding the role of Wnt-signaling on neurodevelopmental and neuropsychiatric disorders is important because of the insight provided on pathogenesis and from the therapeutic perspective; canonical Wnt-signaling is a target of lithium salts, for example. These preliminary studies are currently being expanded to include three additional control and CHD8+/- NPC lines, and whether gene expression changes are affected by lithium.

M60 - EXPRESSION SIGNATURE ASSOCIATED WITH CORTICAL MATURATION AND ITS POTENTIAL ASSOCIATION WITH GENETIC RISK FOR SCHIZOPHRENIA

Background The human neural progenitor cell line CTX0E16 is a robust source of forebrain-like glutamatergic cortical neurons that display intrinsic functional properties (Anderson et al., 2015, Stem Cell Res). This cell line was obtained from the embryonic cortex of a 12-week gestation XY fetus, and conditionally immortalised by ectopic expression of the c-MycER TAM transgene. This construct allows the stem cells to be maintained in a proliferative state under the presence of hydroxytamoxifen (4-OHT), while removal of 4-OHT and growth factors stimulates progenitor differentiation. The cortex is generally smaller in schizophrenia patients, and understanding the global gene expression changes occurring during cortical development, and its relationship to genetic risk for schizophrenia, might reveal clues as to which genetic mechanisms are responsible for mediating the differences observed in patients. Our hypothesis is that neurodevelopmental diseases such as schizophrenia might share a genetic component with processes driving early cortical maturation, which can be modelled in vitro. Methods The CTX0E16 cell line was provided by ReNeuron and total RNA was extracted from proliferating neural progenitor cells (NPCs, n=3) and from cells that underwent a 28-day differentiation protocol (n=3). RNA samples were then processed on Illumina HT12 v4 chips. Raw fluorescence probe intensity values were extracted using GenomeStudio, and were subsequently background-corrected and log-transformed in R using the lumi package. Differentially expressed probes were determined using t-tests and the false-discovery rate of multiple testing correction was applied (q Results We expect to identify and annotate sets of genes involved in cortical maturation that are additionally involved in the aetiology of schizophrenia. Discussion The discussion will focus on the role of cortical maturation as a process that may be involved in schizophrenia aetiology.

High-throughput identification of factors promoting neuronal differentiation of human neural progenitor cells in microscale 3D cell culture.

Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell culture models have been used increasingly for in vitro cell culture because they may better model endogenous tissue environments. However, detailed studies of stem cell differentiation within 3D cultures remain limited, particularly with respect to high-throughput screening. Herein, we demonstrate the use of a microarray chip-based platform to screen, in high-throughput, individual and paired effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures. Dose-response analysis of selected combinations from the initial combinatorial screen revealed that the combined treatment of all-trans retinoic acid (RA) with the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) enhances neurogenesis while simultaneously decreases astrocyte differentiation, whereas the combined treatment of brain-derived neurotrophic factor and the small azide neuropathiazol enhances the differentiation into neurons and astrocytes. Subtype specification analysis of RA- and CHIR-differentiated cultures revealed that enhanced neurogenesis was not biased toward a specific neuronal subtype. Together, these results demonstrate a high-throughput screening platform for rapid evaluation of differentiation conditions in a 3D environment, which will aid the development and application of 3D stem cell culture models.

Characterization of a Human-Specific Tandem Repeat Associated with Bipolar Disorder and Schizophrenia

Bipolar disorder (BD) and schizophrenia (SCZ) are highly heritable diseases that affect more than 3% of individuals worldwide. Genome-wide association studies have strongly and repeatedly linked risk for both of these neuropsychiatric diseases to a 100 kb interval in the third intron of the human calcium channel gene CACNA1C. However, the causative mutation is not yet known. We have identified a human-specific tandem repeat in this region that is composed of 30bp units, often repeated hundreds of times. This large tandem repeat is unstable using standard polymerase chain reaction and bacterial cloning techniques, which may have resulted in its incorrect size in the human reference genome. The large 30-mer repeat region is polymorphic in both size and sequence in human populations. Particular sequence variants of the 30-mer are associated with risk status at several flanking single-nucleotide polymorphisms in the third intron of CACNA1C that have previously been linked to BD and SCZ. The tandem repeat arrays function as enhancers that increase reporter gene expression in a human neural progenitor cell line. Different human arrays vary in the magnitude of enhancer activity, and the 30-mer arrays associated with increased psychiatric disease risk status have decreased enhancer activity. Changes in the structure and sequence of these arrays likely contribute to changes in CACNA1C function during human evolution and may modulate neuropsychiatric disease risk in modern human populations.

Modelling glioma invasion using 3D bioprinting and scaffold-free 3D culture.

Glioma is a highly aggressive form of brain cancer, with some subtypes having 5-year survival rates of less than 5%. Tumour cell invasion into the surrounding parenchyma seems to be the primary driver of these poor outcomes, as most gliomas recur within 2cm of the original surgically-resected tumour. Many current approaches to the development of anticancer therapy attempt to target genetic weaknesses in a particular cancer, but may not take into account the microenvironment experienced by a tumour and the patient-specific genetic differences in susceptibility to treatment. Here we demonstrate the use of complementary approaches, 3D bioprinting and scaffold-free 3D tissue culture, to examine the invasion of glioma cells into neural-like tissue with 3D confocal microscopy. We found that, while both approaches were successful, the use of 3D tissue culture for organoid development offers the advantage of broad accessibility. As a proof-of-concept of our approach, we developed a system in which we could model the invasion of human glioma cells into mouse neural progenitor cell-derived spheroids. We show that we can follow invasion of human tumour cells using cell-tracking dyes and 3D laser scanning confocal microscopy, both in real time and in fixed samples. We validated these results using conventional cryosectioning. Our scaffold-free 3D approach has broad applicability, as we were easily able to examine invasion using different neural progenitor cell lines, thus mimicking differences that might be observed in patient brain tissue. These results, once applied to iPSC-derived cerebral organoids that incorporate the somatic genetic variability of patients, offer the promise of truly personalized treatments for brain cancer.

Human Rett-derived neuronal progenitor cells in 3D graphene scaffold as an in vitro platform to study the effect of electrical stimulation on neuronal differentiation

Studies of electrical stimulation therapies for the treatment of neurological disorders, such as deep brain stimulation, have almost exclusively been performed using animal-models. However, because animal-models can only approximate human brain disorders, these studies should be supplemented with an in vitro human cell-culture based model to substantiate the results of animal-based studies and further investigate therapeutic benefit in humans. This study presents a novel approach to analyze the effect of electrical stimulation on the neurogenesis of patient-induced pluripotent stem cell (iPSC) derived neural progenitor cell (NPC) lines, in vitro using a 3D graphene scaffold system. The iPSC-derived hNPCs used to demonstrate the system were collected from patients with Rett syndrome, a debilitating neurodevelopmental disorder. The graphene scaffold readily supported both the wild-type and Rett NPCs. Electrical stimulation parameters were optimized to accommodate both wild-type and Rett cells. Increased cell maturation and improvements in cell morphology of the Rett cells was observed after electrical stimulation. The results of the pilot study of electrical stimulation to enhance Rett NPCs neurogenesis were promising and support further investigation of the therapy. Overall, this system provides a valuable tool to study electrical stimulation as a potential therapy for neurological disorders using patient-specific cells.

The Effects of Low-Dose Bisphenol A and Bisphenol F on Neural Differentiation of a Fetal Brain-Derived Neural Progenitor Cell Line

Environmental chemicals are known to disrupt the endocrine system in humans and to have adverse effects on several organs including the developing brain. Recent studies indicate that exposure to environmental chemicals during gestation can interfere with neuronal differentiation, subsequently affecting normal brain development in newborns. Xenoestrogen, bisphenol A (BPA), which is widely used in plastic products, is one such chemical. Adverse effects of exposure to BPA during pre- and postnatal periods include the disruption of brain function. However, the effect of BPA on neural differentiation remains unclear. In this study, we explored the effects of BPA or bisphenol F (BPF), an alternative compound for BPA, on neural differentiation using ReNcell, a human fetus-derived neural progenitor cell line. Maintenance in growth factor-free medium initiated the differentiation of ReNcell to neuronal cells including neurons, astrocytes, and oligodendrocytes. We exposed the cells to BPA or BPF for 3 days from the period of initiation and performed real-time PCR for neural markers such as β III-tubulin and glial fibrillary acidic protein (GFAP), and Olig2. The β III-tubulin mRNA level decreased in response to BPA, but not BPF, exposure. We also observed that the number of β III-tubulin-positive cells in the BPA-exposed group was less than that of the control group. On the other hand, there were no changes in the MAP2 mRNA level. These results indicate that BPA disrupts neural differentiation in human-derived neural progenitor cells, potentially disrupting brain development.

Characterization and virus susceptibility of a continuous cell line derived from the brain of Aequidens rivulatus (Günther).

Cell cultures derived from the brain tissues of Aequidens rivulatus (Günther) have been characterized previously. In this study, a continuous cell line ARB8 was further established, and its growth characteristics, transcription and susceptibility to fish viruses-including chum salmon reovirus (CSV), marbled eel infectious pancreative necrosis virus (MEIPNV), grouper nervous necrosis virus (GNNV), giant seaperch iridovirus (GSIV), red seabream iridovirus (RSIV), koi herpesvirus (KHV), herpesvirus anguilla (HVA) and marbled eel polyoma-like virus (MEPyV)-were examined. ARB8 cells that showed epithelioid morphology and were passaged >80 times grew well at temperatures ranging from 25°C to 30°C in L-15 medium containing 5%-15% foetal bovine serum. The cells constitutively transcribed connexion 43, glutamine synthetase, nestin and nkx6-2, which are markers for neural progenitor cells. The cells were highly susceptible to CSV, MEIPNV, GSIV and RSIV and showed the typical cytopathic effect (CPE). However, the cells were resistant to GNNV, KHV, HVA and MEPyV because no significant CPE was noted after infection. Optimal temperatures for virus production ranged from 25°C to 30°C. The results revealed that the neural progenitor cell line ARB8 can potentially serve as a useful tool for investigating fishvirusesand isolating newvirusesin ornamental cichlid fishes.

An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells.

SummaryGrowing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of replicative astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. We evaluated this protocol across 42 NPC lines (derived from 30 individuals). Transcriptomic analysis demonstrated that hiPSC-astrocytes from four individuals are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity, and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our protocol is a reproducible, straightforward (single medium), and rapid (<30 days) method to generate populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders.

Generation and Maintenance of Neural Progenitor Cell Lines Derived from Human Embryonic Stem Cells by Small Molecules

Background: A new research perspective for human neurodevelopment and neurological disease modeling involves the use of human embryonic stem cells (hESC). The development of robust protocols that yield adequate neural cell populations with definite regional identities were the prerequisites for these comparative studies. Materials and Methods: We used small molecules cocktail to generate two neural progenitor cell (NPC) lines from hESCs as following: the first experimental group included Noggin, Dorsomorphin, CHIR99021 and A83-01(NDCA) and the second group composed of Noggin, Dorsomorphin, CHIR99021 and SIS3 (NDCS). To validate our findings, we expanded both cell lines for over 20 passages in vitro and checked for chromosomal stability, as well as expressions of neural and regional identity markers by immunofluorescence staining. Gene expression analysis was quantified by RT-PCR at different passages up to passage 20. Results: Both cell lines proliferated in an adherent culture system in the presence of FGF2. They retained progenitor characteristics of NESTIN, SOX1, and PAX6 protein expression, formed rosette-like structures, and had the high neurogenic capacity. Importantly, the NPC populations in their first 10 passages expressed rostral markers (OTX2 and TH), and the next 10 passages (10-20) changed their specification toward the hindbrain where they expressed HOXA3and HOXB2, which correlated with a normal central nervous system development pattern. Conclusion: These NPCs offer a new system to study human central nervous system development and disease modeling of specific neurodegenerative diseases. [GMJ.2017;6(2):143-56]

Knockdown of the schizophrenia susceptibility gene TCF4 alters gene expression and proliferation of progenitor cells from the developing human neocortex

Common variants in the TCF4 gene are among the most robustly supported genetic risk factors for schizophrenia. Rare TCF4 deletions and loss-of-function point mutations cause Pitt-Hopkins syndrome, a developmental disorder associated with severe intellectual disability. To explore molecular and cellular mechanisms by which TCF4 perturbation could interfere with human cortical development, we experimentally reduced the endogenous expression of TCF4 in a neural progenitor cell line derived from the developing human cerebral cortex using RNA interference. Effects on genome-wide gene expression were assessed by microarray, followed by Gene Ontology and pathway analysis of differentially expressed genes. We tested for genetic association between the set of differentially expressed genes and schizophrenia using genome-wide association study data from the Psychiatric Genomics Consortium and competitive gene set analysis (MAGMA). Effects on cell proliferation were assessed using high content imaging. Genes that were differentially expressed following TCF4 knockdown were highly enriched for involvement in the cell cycle. There was a nonsignificant trend for genetic association between the differentially expressed gene set and schizophrenia. Consistent with the gene expression data, TCF4 knockdown was associated with reduced proliferation of cortical progenitor cells in vitro. A detailed mechanistic explanation of how TCF4 knockdown alters human neural progenitor cell proliferation is not provided by this study. Our data indicate effects of TCF4 perturbation on human cortical progenitor cell proliferation, a process that could contribute to cognitive deficits in individuals with Pitt-Hopkins syndrome and risk for schizophrenia.

Comparison of methods for deriving neural progenitor cells from nonhuman primate embryonic stem cells

Background: Feeder-free monolayer culture and the suspension culture of embryoid bodies (EB) followed by adherent culture, rosette selection, and expansion are 2 methods for deriving neural progenitor cells (NPCs). Direct comparison of these 2 methods has not yet been reported. Objectives: To compare the influence of NPC derivation methods on the properties of NPCs derived from rhesus monkey (Macaca mulatta) embryonic stem cells (rhESCs). Methods: rhESCs were used to derive NPC lines using 2 different methods. EB were produced from a suspension culture of rhESC clumps and rhESCs were cultured in feeder-free monolayers on poly-L-ornithine/laminin coated plates. NPCs were derived by exposure to induction factors. Cell morphology, neural and nonneural lineage markers were evaluated. We measured the expression of nuclear receptor tailless (TLX), which acts as repressor of glial fibrillary acidic protein (GFAP) expression. Results: NPCs were successful derived using either method, with homogenous populations based on the expression of nestin (>97%) and Pax6 (>99%) as shown by flow cytometry. No significant difference in NPC specific markers or ability to differentiate into neurons in vitro was found between the methods. However, the expression of GFAP was >400-fold higher in cells produced by the feeder-free method. This distinction was consistent with the lower expression of TLX. Conclusions: NPCs derived by feeder-free and EB methods share similar morphology and properties. The elevated expression of GFAP and reduced expression of TLX in NPCs derived using the feeder-free method may explain their greater heterogeneity and tendency to differentiate toward cells of an astrocyte lineage. Keywords: Embryonic stem cells, neural progenitor cells, neural progenitor cell derivation, nonhuman primate,TLX

USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors

USP9X, is highly expressed in neural progenitors and, essential for neural development in mice. In humans, mutations in USP9X are associated with neurodevelopmental disorders. To understand USP9X’s role in neural progenitors, we studied the effects of altering its expression in both the human neural progenitor cell line, ReNcell VM, as well as neural stem and progenitor cells derived from Nestin-cre conditionally deleted Usp9x mice. Decreasing USP9X resulted in ReNcell VM cells arresting in G0 cell cycle phase, with a concomitant decrease in mTORC1 signalling, a major regulator of G0/G1 cell cycle progression. Decreased mTORC1 signalling was also observed in Usp9x-null neurospheres and embryonic mouse brains. Further analyses revealed, (i) the canonical mTORC1 protein, RAPTOR, physically associates with Usp9x in embryonic brains, (ii) RAPTOR protein level is directly proportional to USP9X, in both loss- and gain-of-function experiments in cultured cells and, (iii) USP9X deubiquitlyating activity opposes the proteasomal degradation of RAPTOR. EdU incorporation assays confirmed Usp9x maintains the proliferation of neural progenitors similar to Raptor-null and rapamycin-treated neurospheres. Interestingly, loss of Usp9x increased the number of sphere-forming cells consistent with enhanced neural stem cell self-renewal. To our knowledge, USP9X is the first deubiquitylating enzyme shown to stabilize RAPTOR.

Divergent Levels of Marker Chromosomes in an hiPSC-Based Model ofPsychosis.

SummaryIn the process of generating presumably clonal human induced pluripotent stem cells (hiPSCs) from two carriers of a complex structural rearrangement, each having a psychotic disorder, we also serendipitously generated isogenic non-carrier control hiPSCs, finding that the rearrangement occurs as an extrachromosomal marker (mar) element. All confirmed carrier hiPSCs and differentiated neural progenitor cell lines were found to be mosaic. We caution that mar elements may be difficult to functionally evaluate in hiPSC cultures using currently available methods, as it is difficult to distinguish cells with and without mar elements in live mosaic cultures.

Altered mRNA Expression and Cell Membrane Potential in the Differentiated C17.2 Cell Model as Indicators of Acute Neurotoxicity

Abstract Using general cytotoxicity assays in combination with in vitro tests for organ-specific toxicity has been proposed as an alternative approach to animal tests for estimation of acute systemic toxicity. Here, we present the C17.2 neural progenitor cell line as an option for estimation of acute neurotoxicity. The C17.2 cells were differentiated for 6 days in serum-free N2 medium with brain-derived neurotrophic factor and nerve growth factor to a mixed culture of neurons and astrocytes. The cells were then exposed to noncytotoxic concentrations of acetylsalicylic acid, atropine, digoxin, ethanol, nicotine, or strychnine for 48 hours and the mRNA levels of glial fibrillary acidic protein, βIII-tubulin, and heat shock protein 32 were analyzed as biomarkers for astrocytes, neurons, and cellular stress respectively. As a functional endpoint, the cell membrane potential (CMP) was monitored after acute addition of each compound to the differentiated C17.2 cells, by using the fluorescent FLIPR® membrane pot...