Human Cell-based Model Research Articles

Isolation of human testicular cells and co-culture with embryonic stem cells.

Our overall goal is to create a three-dimensional human cell-based testicular model for toxicological and spermatogenesis studies. Methods to purify the major somatic testicular cells, namely Leydig cells (LCs), peritubular myoid cells (PCs) and Sertoli cells (SCs), from rats, mice and guinea pigs have been reported. In humans, the isolation of populations enriched for primary LCs, PCs or SCs also have described. One objective of this study was to determine if populations of cells enriched for all three of these cell types can be isolated from testes of single human donors, and we were successful in doing so from testes of three donors. Testes tissues were enzymatically digested, gravity sedimented and Percoll filtered to isolate populations enriched for LCs, PCs and SCs. LCs and PCs were identified by colorimetric detection of the expression of prototypical enzymes. Division of PCs and SCs in culture has been reported. We observed that primary human LCs could divide in culture by incorporation of 5-ethynyl-2'-deoxyuridine. SCs were identified and their functionality was demonstrated by the formation of tight junctions as shown by the expression of tight junction proteins, increased transepithelial electrical resistance, polarized secretion of biomolecules and inhibition of lucifer yellow penetration. Furthermore, we found that human SC feeder layers could facilitate germ cell progression of human embryonic stem cells (hESCs) by microarray analysis of gene expression.

Investigating the effects of Orexin-A on thermogenesis in human deep neck brown adipose tissue.

Despite successful preclinical testing, 85% of early clinical trials for novel drugs fail. Most futilities originate from molecular mechanisms of the drug(s) tested. It is critically important to develop validated human cell-based model systems in which animal-based research can be translated in order to complement the preclinical in vivo findings prior to implementation of a clinical trial. Obesity is associated with reduced circulating levels of Orexin-A (OX-A) in humans. OX-A increases thermogenesis in rodent brown adipose tissue (AT), yet this phenomenon has not been explored in humans. We established a cell-based model system of human brown and white adipocytes and tested the effects of OX-A on thermogenesis. Contrary to published in vivo and in vitro reports in rodents, OX-A treatment alone or in combination with an adrenergic stimulus did neither enhance thermogenesis nor its related transcriptional program in a human in vitro model of brown adipocytes or AT explants. Translating preclinical findings in human model systems poses a challenge that must be overcome for the development of effective therapeutic compounds and targets.

Abstract 1936: Epigenetic challenges in derivation of the first cell-based model of Beckwith-Wiedemann Syndrome

Abstract Beckwith-Wiedemann Syndrome (BWS) is a cancer predisposition syndrome that affects at least 1 in 10,500 children. Up to 25% of children with BWS develop tumors, primarily Wilms tumor and hepatoblastoma. BWS is due to genetic or epigenetic changes that affect imprinted loci on chromosome 11 and these same changes are also found in other types of cancer. There are no cell-based models of BWS and most mouse models do not recapitulate the tumor phenotype. To understand more about the mechanisms leading to tumor formation in BWS, we developed the first human cell-based model of BWS. Human induced pluripotent stem cell (iPSC) models are commonly used to study disease mechanisms in tissue types that are not normally accessible for study. In the case of BWS, we plan to use such models to study how the genetic and epigenetic changes in BWS lead to tumor formation in hepatic and renal cells. Using skin fibroblasts from four BWS patients, we derived the first iPSC models of BWS. Prior to iPSC derivation, we characterized different tissues available from these four patients, and demonstrated genetic mosaicism in different tissue types, including blood, skin, and pancreas. During the derivation process, we demonstrated that both normal and BWS iPSC lines could be derived from the same patient fibroblast sample and the number of clones of each type from each sample approximated the initial level of mosaicism in the original sample. For each patient, these lines are isogenic except for the BWS critical region. The BWS and isogenic normal iPSCs were characterized for pluripotency markers and demonstrated to have normal karyotypes. Following this analysis, BWS and isogenic normal lines were characterized extensively for DNA methylation at specific imprinted loci in both early passage and extended culture. Methylation analysis was performed by both pyrosequencing and COBRA assays. Methylation was maintained at some imprinted loci but not at others in extended culture. Importantly, relatively stable methylation levels were observed at the BWS critical imprinted regions (H19/IGF2 and KvDMR), regardless of methods of reprogramming, indicating a relatively stable state of DNA methylation in this region. Additionally, normal methylation was seen at the SNRPN locus. In contrast, another imprinted locus, IG/MEG3, displayed abnormal hypermethylation in iPSCs. These data indicate that reprogramming and extended culture of iPSCs can affect stability of DNA methylation at certain imprinted loci. Therefore caution should be used in interpreting studies using iPSCs as these aberrant methylation states at imprinted loci can affect the downstream functionality of iPSC models. BWS iPSCs and isogenic controls with normal methylation will be used for further study of the mechanism of tumor formation in BWS. Note: This abstract was not presented at the meeting. Citation Format: Stella K. Hur, Joanne L. Thorvaldsen, Suhee Chang, Carolyn Lye, Alice Yu, Monserrat C. Anguera, Marisa S. Bartolomei, Jennifer M. Kalish. Epigenetic challenges in derivation of the first cell-based model of Beckwith-Wiedemann Syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1936. doi:10.1158/1538-7445.AM2017-1936

Luteolin attenuates interleukin-6-mediated astrogliosis in human iPSC-derived neural aggregates: A candidate preventive substance for maternal immune activation-induced abnormalities.

Maternal infection during pregnancy increases the risk of neurodevelopmental conditions such as autism spectrum disorders and schizophrenia in offspring. Several previous animal studies have indicated that maternal immune activation (MIA), rather than a specific pathogen, alters fetal brain development. Among them, prenatal exposure to interleukin-6 (IL-6) has been associated with behavioral and neuropathological abnormalities, though such findings remain to be elucidated in humans. We developed a human cell-based model of MIA by exposing human induced pluripotent stem cells (hiPSCs)-derived neural aggregates to IL-6 and investigated whether luteolin-a naturally occurring flavonoid found in edible plants-could prevent MIA-induced abnormalities. We generated neural aggregates from hiPSCs using the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) method, following which aggregates were cultured in suspension. We then exposed the aggregates to IL-6 (100ng/ml) for 24h at day 51. Transient IL-6 exposure significantly increased the area ratio of astrocytes (GFAP-positive area ratio) and decreased the area ratio of early-born neurons (TBR1-positive or CTIP2-positive area ratio) relative to controls. In addition, western blot analysis revealed that levels of phosphorylated STAT3 were significantly elevated in IL-6-exposed neural aggregates. Luteolin treatment inhibited STAT3 phosphorylation and counteracted IL-6-mediated increases of GFAP-positive cells and reductions of TBR1-positive and CTIP2-positive cells. Our observations suggest that the flavonoid luteolin may attenuate or prevent MIA-induced neural abnormalities. As we observed increased apoptosis at high concentrations of luteolin, further studies are required to determine the optimal intake dosage and duration for pregnant women.

A New Assessment of the Prevalence and Risk Factors of Liver Abnormalities in Hyperthyroidism

Clinical ThyroidologyVol. 29, No. 5 SummariesA New Assessment of the Prevalence and Risk Factors of Liver Abnormalities in HyperthyroidismJacques OrgiazziJacques OrgiazziSearch for more papers by this authorPublished Online:1 May 2017https://doi.org/10.1089/ct.2017;29.186-188AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byEstablishment of a human embryonic stem cell-based liver differentiation model for hepatotoxicity evaluationsEcotoxicology and Environmental Safety, Vol. 174 Volume 29Issue 5May 2017 InformationCopyright 2017 American Thyroid Association, Inc.To cite this article:Jacques Orgiazzi.A New Assessment of the Prevalence and Risk Factors of Liver Abnormalities in Hyperthyroidism.Clinical Thyroidology.May 2017.186-188.http://doi.org/10.1089/ct.2017;29.186-188Published in Volume: 29 Issue 5: May 1, 2017PDF download

The impact of Zika virus infection on human neuroblastoma (SH-SY5Y) cell line.

An increase in Zika virus (ZIKV) epidemic during the last decade has become a major global concern as the virus affects both newborns and adult humans. Earlier studies have shown the impact of ZIKV infection in developing human foetus. However, effective in vitro model of target cells for studying the ZIKV infection in adult human neurons is not available. This study aimed to establish the use of human neuroblastoma cell line (SH-SY5Y) for studying an infection of ZIKV in vitro. ZIKV growth kinetics, viral toxicity, and SH-SY5Y cell vialibity were determined after ZIKV infection in SH-SY5Y cells in vitro. ZIKV-infected SH-SY5Y cells were morphologically analysed and compared with nonhuman primate Vero cells. Furthermore, the susceptibility of SH-SY5Y cells to ZIKV infection was also determined. The results showed that ZIKV efficiently infects SH-SY5Y cell lines in vitro. Gradual changes of several cellular homeostasis parameters including cell viability, cytotoxicity, and cell morphology were observed in ZIKVinfected SH-SY5Y cells when compared to mock-treated or non-human primate cells. Interestingly, ZIKV particles were detected in the nucleoplasmic compartment of the infected SH-SY5Y cells. The results suggest that ZIKV particle can be detected in the nucleoplasmic compartment of the infected SH-SY5Y cells beside the known viral replicating cytoplasmic area. Hence, SH-SY5Y cells can be used as an in vitro adult human neuronal cell-based model, for further elucidating the ZIKV biology, and highlight other possible significance of Zika virus distribution through nuclear localization, which may correlate to the neuropathological defects in ZIKV-infected adult humans.

A Human Pluripotent Stem Cell Model of Facioscapulohumeral Muscular Dystrophy-Affected Skeletal Muscles.

: Facioscapulohumeral muscular dystrophy (FSHD) represents a major unmet clinical need arising from the progressive weakness and atrophy of skeletal muscles. The dearth of adequate experimental models has severely hampered our understanding of the disease. To date, no treatment is available for FSHD. Human embryonic stem cells (hESCs) potentially represent a renewable source of skeletal muscle cells (SkMCs) and provide an alternative to invasive patient biopsies. We developed a scalable monolayer system to differentiate hESCs into mature SkMCs within 26 days, without cell sorting or genetic manipulation. Here we show that SkMCs derived from FSHD1-affected hESC lines exclusively express the FSHD pathogenic marker double homeobox 4 and exhibit some of the defects reported in FSHD. FSHD1 myotubes are thinner when compared with unaffected and Becker muscular dystrophy myotubes, and differentially regulate genes involved in cell cycle control, oxidative stress response, and cell adhesion. This cellular model will be a powerful tool for studying FSHD and will ultimately assist in the development of effective treatments for muscular dystrophies. This work describes an efficient and highly scalable monolayer system to differentiate human pluripotent stem cells (hPSCs) into skeletal muscle cells (SkMCs) and demonstrates disease-specific phenotypes in SkMCs derived from both embryonic and induced hPSCs affected with facioscapulohumeral muscular dystrophy. This study represents the first human stem cell-based cellular model for a muscular dystrophy that is suitable for high-throughput screening and drug development.

Role of the YAP Oncoprotein in Priming Ras-Driven Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), a cancer characterized by features of skeletal muscle histogenesis, is the most common soft tissue sarcoma of childhood and adolescence. Survival for high-risk groups is less than 30% at 5 years. RMS also occurs during adulthood, with a lower incidence but higher mortality. Recently, mutational profiling has revealed a correlation between activating Ras mutations in the embryonal (eRMS) and pleomorphic (pRMS) histologic variants of RMS, and a poorer outcome for those patients. Independently, the YAP transcriptional coactivator, an oncoprotein kept in check by the Hippo tumor suppressor pathway, is upregulated in eRMS. Here we show that YAP promotes cell proliferation and antagonizes apoptosis and myogenic differentiation of human RMS cells bearing oncogenic Ras mutations in cell culture studies in vitro and in murine xenografts in vivo. Pharmacologic inhibition of YAP by the benzoporphyrin derivative verteporfin decreased cell proliferation and tumor growth in vivo. To interrogate the temporal contribution of YAP in eRMS tumorigenesis, we used a primary human cell-based genetic model of Ras-driven RMS. Constitutively active YAP functioned as an early genetic lesion, permitting bypass of senescence and priming myoblasts to tolerate subsequent expression of hTERT and oncogenic Ras, which were necessary and sufficient to generate murine xenograft tumors mimicking RMS in vivo. This work provides evidence for cooperation between YAP and oncogenic Ras in RMS tumorigenesis, laying the foundation for preclinical co-targeting of these pathways.

Abstract 3230: Human stem cell-based model of MYCN-driven neuroblastoma

Abstract Neuroblastoma (NB), a disease of neural crest (NC) origin, is the most common extracranial solid tumor in childhood. High-risk NB patients represent the subgroup with the worst prognosis and frequently harbor amplification of MYCN. Due to its biochemical structure as a transcription factor, MYCN has been difficult to target directly with small molecules. Alternatively, identification of cooperating partners of MYCN-induced tumorigenesis can reveal a more therapeutically viable target, such as anaplastic lymphoma kinase (ALK). While genetically engineered mouse models (GEMMs) of NB driven by MYCN and ALK exist, recent studies have found significant differences between mouse models of disease and the human tumors they are intended to represent. In support of this, mouse and human NC cells differ in development and marker expression, and the genetic requirement for transformation of human cells has been shown to be more complex than mouse cells, suggesting that a human cell-based NB model would be more relevant. To develop a human cell-based model of NB, we started with a normal human induced pluripotent stem (iPS) cell line derived integration-free from a healthy adult and transduced empty vector, ALK F1174L (active mutant), doxycycline-inducible MYCN (DOX-MYCN), or ALK F1174L/DOX-MYCN. These iPS cells were differentiated towards NC cells and subsequently implanted orthotopically into renal capsules of mice fed on dox chow. Within 3 months, 60% of mice developed tumors with ALK F1174L/DOX-MYCN, 10% with DOX-MYCN, and 0% with both empty vector and ALK F1174L alone. Tumors were transplantable and demonstrated histologic characteristics consistent with NB, including morphology and expression markers. Thus, we are demonstrating the first, to our knowledge, human stem cell-based model of NB. We are using this system to further investigate similarities and differences with GEMMs of NB, test and establish novel candidate drivers of NB, and evaluate potential therapeutic options. Citation Format: Miller Huang, Lauren K. McHenry, Matthew L. Miller, Grace E. Kim, Branden S. Moriarity, Yuichiro Miyaoka, Marianne E. Bronner, David A. Largaespada, Bruce R. Conklin, Hanlee P. Ji, John M. Maris, Katherine K. Matthay, William A. Weiss. Human stem cell-based model of MYCN-driven neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3230. doi:10.1158/1538-7445.AM2015-3230

The EpiOcular Eye Irritation Test (EIT) for hazard identification and labelling of eye irritating chemicals: protocol optimisation for solid materials and the results after extended shipment.

The 7th Amendment to the EU Cosmetics Directive and the EU REACH Regulation have reinforced the need for in vitro ocular test methods. Validated in vitro ocular toxicity tests that can predict the human response to chemicals, cosmetics and other consumer products are required for the safety assessment of materials that intentionally, or inadvertently, come into contact with the eye. The EpiOcular Eye Irritation Test (EIT), which uses the normal human cell-based EpiOcular™ tissue model, was developed to address this need. The EpiOcular-EIT is able to discriminate, with high sensitivity and accuracy, between ocular irritant/corrosive materials and those that require no labelling. Although the original EpiOcular-EIT protocol was successfully pre-validated in an international, multicentre study sponsored by COLIPA (the predecessor to Cosmetics Europe), data from two larger studies (the EURL ECVAM-COLIPA validation study and an independent in-house validation at BASF SE) resulted in a sensitivity for the protocol for solids that was below the acceptance criteria set by the Validation Management Group (VMG) for eye irritation, and indicated the need for improvement of the assay's sensitivity for solids. By increasing the exposure time for solid materials from 90 minutes to 6 hours, the optimised EpiOcular-EIT protocol achieved 100% sensitivity, 68.4% specificity and 84.6% accuracy, thereby meeting all the acceptance criteria set by the VMG. In addition, to satisfy the needs of Japan and the Pacific region, the EpiOcular-EIT method was evaluated for its performance after extended shipment and storage of the tissues (4-5 days), and it was confirmed that the assay performs with similar levels of sensitivity, specificity and reproducibility in these circumstances.

Elucidating Mechanisms of Toxicity Using Phenotypic Data from Primary Human Cell Systems—A Chemical Biology Approach for Thrombosis-Related Side Effects

Here we describe a chemical biology approach for elucidating potential toxicity mechanisms for thrombosis-related side effects. This work takes advantage of a large chemical biology data set comprising the effects of known, well-characterized reference agents on the cell surface levels of tissue factor (TF) in a primary human endothelial cell-based model of vascular inflammation, the BioMAP® 3C system. In previous work with the Environmental Protection Agency (EPA) for the ToxCast™ program, aryl hydrocarbon receptor (AhR) agonists and estrogen receptor (ER) antagonists were found to share an usual activity, that of increasing TF levels in this system. Since human exposure to compounds in both chemical classes is associated with increased incidence of thrombosis-related side effects, we expanded this analysis with a large number of well-characterized reference compounds in order to better understand the underlying mechanisms. As a result, mechanisms for increasing (AhR, histamine H1 receptor, histone deacetylase or HDAC, hsp90, nuclear factor kappa B or NFκB, MEK, oncostatin M receptor, Jak kinase, and p38 MAPK) and decreasing (vacuolar ATPase or V-ATPase) and mTOR) TF expression levels were uncovered. These data identify the nutrient, lipid, bacterial, and hypoxia sensing functions of autophagy as potential key regulatory points controlling cell surface TF levels in endothelial cells and support the mechanistic hypothesis that these functions are associated with thrombosis-related side effects in vivo.

MiRNA expression profiling in a human stem cell-based model as a tool for developmental neurotoxicity testing

The main aim of this study was to evaluate whether microRNA (miRNA) profiling could be a useful tool for in vitro developmental neurotoxicity (DNT) testing. Therefore, to identify the possible DNT biomarkers among miRNAs, we have studied the changes in miRNA expressions in a mixed neuronal/glial culture derived from carcinoma pluripotent stem cells (NT2 cell line) after exposure to methyl mercury chloride (MeHgCl) during the process of neuronal differentiation (2-36days in vitro (DIV1)). The neuronal differentiation triggered by exposure to retinoic acid (RA) was characterized in the control culture by mRNA expression analysis of neuronal specific markers such as MAP2, NF-200, Tubulin βIII, MAPT-tau, synaptophysin as well as excitatory (NMDA, AMPA) and inhibitory (GABA) receptors. The results obtained from the miRNA expression analysis have identified the presence of a miRNA signature which is specific for neural differentiation in the control culture and another for the response to MeHgCl-induced toxicity. In differentiated neuronal control cultures, we observed the downregulation of the stemness phenotype-linked miR-302 cluster and the overexpression of several miRNAs specific for neuronal differentiation (e.g. let-7, miR-125b and miR-132). In the cultures exposed to MeHgCl (400nM), we observed an overexpression of a signature composed of five miRNAs (miR-302b, miR-367, miR-372, miR-196b and miR-141) that are known to be involved in the regulation of developmental processes and cellular stress response mechanisms. Using gene ontology term and pathway enrichment analysis of the validated targets of the miRNAs deregulated by the toxic treatment, the possible effect of MeHgCl exposure on signalling pathways involved in axon guidance and learning and memory processes was revealed. The obtained data suggest that miRNA profiling could provide simplified functional evaluation of the toxicity pathways involved in developmental neurotoxicity in comparison with the transcriptomics studies.

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: Effects of methylmercury, lead and aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.

A Comprehensive Small Interfering RNA Screen Identifies Signaling Pathways Required for Gephyrin Clustering

The postsynaptic scaffold protein gephyrin is clustered at inhibitory synapses and serves for the stabilization of GABA(A) receptors. Here, a comprehensive kinome-wide siRNA screen in a human HeLa cell-based model for gephyrin clustering was used to identify candidate protein kinases implicated in the stabilization of gephyrin clusters. As a result, 12 hits were identified including FGFR1 (FGF receptor 1), TrkB, and TrkC as well as components of the MAPK and mammalian target of rapamycin (mTOR) pathways. For confirmation, the impact of these hits on gephyrin clustering was analyzed in rat primary hippocampal neurons. We found that brain-derived neurotrophic factor (BDNF) acts on gephyrin clustering through MAPK signaling, and this process may be controlled by the MAPK signaling antagonist sprouty2. BDNF signaling through phosphatidylinositol 3-kinase (PI3K)-Akt also activates mTOR and represses GSK3β, which was previously shown to reduce gephyrin clustering. Gephyrin is associated with inactive mTOR and becomes released upon BDNF-dependent mTOR activation. In primary neurons, a reduction in the number of gephyrin clusters due to manipulation of the BDNF-mTOR signaling is associated with reduced GABA(A) receptor clustering, suggesting functional impairment of GABA signaling. Accordingly, application of the mTOR antagonist rapamycin leads to disinhibition of neuronal networks as measured on microelectrode arrays. In conclusion, we provide evidence that BDNF regulates gephyrin clustering via MAPK as well as PI3K-Akt-mTOR signaling.

Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome

Exposure to the antiepileptic drug valproic acid (VPA) during gestation causes neurofunctional and anatomic deficits in later life. At present, there are little human data on how early neural development is affected by chemicals. We used human embryonic stem cells, differentiating to neuroectodermal precursors, as a model to investigate the modes of action of VPA. Microarray expression profiling, qPCR of specific marker genes, immunostaining and the expression of green fluorescent protein under the control of the promoter of the canonical neural precursor cell marker HES5 were used as readouts. Exposure to VPA resulted in distorted marker gene expression, characterized by a relative increase in NANOG and OCT4 and a reduction in PAX6. A similar response pattern was observed with trichostatin A, a potent and specific histone deacetylase inhibitor (HDACi), but not with several other toxicants. Differentiation markers were disturbed by prolonged, but not by acute treatment with HDACi, and the strongest disturbance of differentiation was observed by toxicant exposure during early neural fate decision. The increased acetylation of histones observed in the presence of HDACi may explain the up-regulation of some genes. However, to understand the down-regulation of PAX6 and the overall complex transcript changes, we examined further epigenetic markers. Alterations in the methylation of lysines 4 and 27 of histone H3 were detected in the promoter region of PAX6 and OCT4. The changes in these activating and silencing histone marks provide a more general mechanistic rational for the regulation of developmentally important genes at non-cytotoxic drug concentrations.

Anti-Aβ drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease.

BackgroundAlzheimer's disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline during middle to late adult life. The AD brain is characterized by deposition of amyloid β peptide (Aβ), which is produced from amyloid precursor protein by β- and γ-secretase (presenilin complex)-mediated sequential cleavage. Induced pluripotent stem (iPS) cells potentially provide an opportunity to generate a human cell-based model of AD that would be crucial for drug discovery as well as for investigating mechanisms of the disease.Methodology/Principal FindingsWe differentiated human iPS (hiPS) cells into neuronal cells expressing the forebrain marker, Foxg1, and the neocortical markers, Cux1, Satb2, Ctip2, and Tbr1. The iPS cell-derived neuronal cells also expressed amyloid precursor protein, β-secretase, and γ-secretase components, and were capable of secreting Aβ into the conditioned media. Aβ production was inhibited by β-secretase inhibitor, γ-secretase inhibitor (GSI), and an NSAID; however, there were different susceptibilities to all three drugs between early and late differentiation stages. At the early differentiation stage, GSI treatment caused a fast increase at lower dose (Aβ surge) and drastic decline of Aβ production.Conclusions/SignificanceThese results indicate that the hiPS cell-derived neuronal cells express functional β- and γ-secretases involved in Aβ production; however, anti-Aβ drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation.

Development of the EpiOcular™ Eye Irritation Test for Hazard Identification and Labelling of Eye Irritating Chemicals in Response to the Requirements of the EU Cosmetics Directive and REACH Legislation

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular(TM) eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients (r²) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular's long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants.

In vitro human cell-based model of the human blood brain barrier (BBB)

In vitro human cell-based model of the human blood brain barrier (BBB)

A Human Stem Cell-Based Model for Identifying Adverse Effects of Organic and Inorganic Chemicals on the Developing Nervous System

The aim of our study was to investigate whether a human neural stem cell line derived from umbilical cord blood (HUCB-NSC) can serve as a reliable test model for developmental neurotoxicity (DNT). We assessed the sensitivity of HUCB-NSCs at different developmental stages to a panel of neurotoxic (sodium tellurite, methylmercury chloride, cadmium chloride, chlorpyrifos, and L-glutamate) and non-neurotoxic (acetaminophen, theophylline, and D-glutamate) compounds. In addition, we investigated the effect of some compounds on key neurodevelopmental processes like cell proliferation, apoptotic cell death, and neuronal and glial differentiation. Less differentiated HUCB-NSCs were generally more sensitive to neurotoxicants, with the notable exception of L-glutamate, which showed a higher toxicity to later stages. The relative potencies of the compounds were: cadmium chloride > methylmercury chloride >> chlorpyrifos >> L-glutamate. Fifty nanomolar methylmercury chloride (MeHgCl) inhibited proliferation and induced apoptosis in early-stage cells. At the differentiated stage, 1 muM MeHgCl induced selective loss of S100 beta-expressing astrocytic cells. One millimolar L-glutamate did not influence the early stages of HUCB-NSC development, but it affected late stages of neuronal differentiation. A valuable system for in vitro DNT assessment should be able to discriminate between neurotoxic and non-neurotoxic compounds and show different susceptibilities to chemicals according to developmental stage and cell lineage. Although not exhaustive, this work shows that the HUCB-NSC model fulfils these criteria and may serve as a human in vitro model for DNT priority setting.

Stem Cell-Based Models and Therapies for Neurodegenerative Diseases

Multiple neurodegenerative disorders typically result from irrevocable damage and improper functioning of specialized neuronal cells or populations of neuronal cells. These disorders have the potential to contribute to an already overburdened health care system unless the progression of neurodegeneration can be altered. Progress in understanding neurodegenerative cell biology has been hampered by a lack of predictive and, some would claim, relevant cellular models. Additionally, the research needed to develop new drugs and determine methods for repair or replacement of damaged neurons is severely hampered by the lack of an adequate in vitro human neuron cell-based model. In this context, pluripotent stem cells and neural progenitors and their properties including unlimited proliferation, plasticity to generate other cell types, and a readily available source of cells--pose an excellent alternative to ex vivo primary cultures or established immortalized cell lines in contributing to our understanding of neurodegenerative cell biology and our ability to analyze the therapeutic or cytotoxic effects of chemicals, drugs, and xenobiotics. Many questions that define the underlying "genesis" of the neuronal death in these disorders also remain unanswered, with evidence suggesting a key role for mitochondrial dysfunction. The assessment of stem cells, neural progenitors, and engineered adult cells can provide useful insights into neuronal development and neurodegenerative processes. Finally, the potential for a combination of cell- and gene-based therapeutics for neurodegenerative disorders is also discussed.