Leonora Buzanska

Workgroup Report: Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity into International Hazard and Risk Assessment Strategies

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19–21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work.

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 μM MeHgCl induced selective loss of S100β‐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 CELLS 2009;27:2591–2601

Micro-stamped surfaces for the patterned growth of neural stem cells

We present a method for patterning neural stem cells based on pre-patterning polypeptides on a cell-repellent surface (poly(ethylene) oxide-like, PEO-like, plasma-deposited films). The method ensures cell attachment and stability for several weeks, as well as it allows cell migration and differentiation. Various patterns of approximately 1 nm thick cell adhesive poly-L-lysine (PLL) have been created on a cell-repellent PEO-like matrix by microcontact printing using different array configurations and printing conditions. The cell-repellent property of PEO-like film determined the confinement of the cells on the printed patterns. Optimization of the printing method showed that the most homogeneous patterns over large areas were obtained using PLL diluted in carbonate buffer (100mM) at pH 8.4. Neural stem cells cultured on the PLL patterns in low serum and in differentiating medium over 20 days exhibited a good confinement to the polypeptide domains. The number of cells attached increased linearly with the micro-stamped PLL area. The cells were able to extend random axon-like projections to the outside of the patterns and presented high amount of ramifications when cultured in differentiating medium. Migration and axon-like outgrowth have been successfully guided by means of an interconnected squares configuration. The surfaces are suitable for controlling the patterning of stem cells and provide a platform for the assessment of the way how different cell arrangements and culture conditions influence cell interactions and cell developmental processes.

In vitro developmental neurotoxicity (DNT) testing: relevant models and endpoints.

Environmental chemicals have a potential impact on childrens health as the developing brain is much more vulnerable to injury caused by different classes of chemicals than the adult brain. This vulnerability is partly due to the fact that very complex processes of cell development and maturation take place within a tightly controlled time frame. So different stages of brain development are susceptible to toxic effects at different time points. Additionally the adult brain is well protected against chemicals by the blood brain barrier (BBB) whereas the placenta only partially protects against harmful chemical exposure. Many metals easily cross the placenta and BBB barrier since even after the birth BBB is not entirely differentiated (until about 6 months after birth). Additionally, the susceptibility of infants and children is due to increased exposure, augmented absorption rates, and less efficient ability of defense mechanism in comparison to adults. The In Vitro Session during the 12th International Neurotoxicology Association meeting (Jerusalem, June, 2009) provided the opportunity to discuss the new challenges that have to be faced to create new type of safety assessments for regulatory requirements. The integration of various tests into testing strategies as well as combination of information-rich approaches with bioinformatics was discussed. Furthermore relevant models and endpoints for developmental neurotoxicity (DNT) evaluation using in vitro approach were presented. The primary neuronal cultures of cerebellar granule cells (CGCs) as well as 3D aggregate model and the possible application of human embryonic and adult stem cells was discussed pointing out the potential of these models to be used for DNT testing. The presented systems are relevant for DNT evaluation as the key processes of brain development such cell proliferation, migration and neuronal/glial differentiation are present. Furthermore, emerging technologies such as gene expression, electrical activity measurements and metabonomics have been identified as promising tools. In a combination with other assays the in vitro approach could be included into a DNT intelligent testing strategy to speed up the process of DNT evaluation mainly by initial prioritization of chemicals with DNT potential for further testing.

Relevance of in vitro neurotoxicity testing for regulatory requirements: Challenges to be considered

The current testing requirements for both adult and developmental neurotoxicity evaluation are based on in vivo animal models and the neurotoxic potency of compounds is mainly determined by neurobehavioural and neuropathological effects. In vitro studies are considered complementary to animal tests because they provide an understanding of the molecular/cellular mechanisms involved in neurotoxicity. However, the selection of relevant in vitro neuronal/glial specific endpoints applied to various neuronal cellular models should be done in a careful way to build reliable and feasible testing strategies since usually these endpoints have to be tested in various complementary in vitro systems. The requirements for applying a more complex test strategy where toxicokinetic aspects are included together with different tools to compensate for the lack of in vitro metabolic competence are discussed. Taking into consideration the recent European Commission chemical legislation concerning registration, evaluation and authorisation of chemicals (REACH) it has become a priority to develop new intelligent testing strategies integrating computational models and in vitro assays based on cell culture models and endpoints that are amenable for adaptation to high throughput screening to be able to test a large number of chemicals.

Neurogenic potential of human umbilical cord blood: Neural-like stem cells depend on previous long-term culture conditions

In vitro studies conducted by our research group documented that neural progenitor cells can be selected from human umbilical cord blood (HUCB‐NPs). Due to further expansion of these cells we have established the first human umbilical cord blood‐derived neural‐like stem cell line (HUCB‐NSC) growing in serum‐free (SF) or low‐serum (LS) medium for over 3 years. The purpose of the study was to evaluate the neurogenic potential of HUCB‐NSCs cultured in SF and LS condition in different in vitro settings before transplantation. We have shown that the number of cells attaining neuronal features was significantly higher for cultures expanded in LS than in SF condition. Moreover, the presence of neuromorphogens, cultured rat astrocytes or hippocampal slices promoted further differentiation of HUCB‐NSCs into neural lineage much more effectively when the cells had derived from LS cultures. The highest response was observed in the case of co‐cultures with rat primary astrocytes as well as hippocampal organotypic slices. However, the LS cells co‐cultured with hippocampal slices expressed exclusively a set of early and late neuronal markers whereas no detection of cells with glial‐specific markers was possible. In conclusion, certain level of stem/progenitor cell commitment is important for optimal response of HUCB‐NSC on the neurogenic signals provided by surrounding environment in vitro.

Neuronal Differentiation of Human Umbilical Cord Blood Neural Stem-Like Cell Line

The expanding population of neural stem/progenitor cells can be selected from human cord blood nonhematopoietic (CD34-negative) mononuclear fraction. Due to repeated expansion and selection of these cells we have established the first clonogenic, nonimmortalized human umbilical cord blood neural stem-like cell (HUCB-NSC) line. This line can be maintained at different stages of neural progenitor development by the presence of trophic factors, mitogens and neuromorphogens in culture media. Neurogenic potential of HUCB-NSC was established for serum-free and low-serum cultured cells. Commitment of HUCB-NSC by serum was shown to be important for the optimal response to the signals provided by surrounding environment in vitro. Enhanced neuronal differentiation induced by dBcAMP treatment was accompanied by expression of several functional proteins including glutamatergic, GABAergic, dopamine, serotonin and acetylcholine receptors, which was shown by microarray, immunocytochemistry and electrophysiology. Electrophysiological studies, whole-cell patch-clamp recordings, revealed in differentiated HUCB-NSC two types of voltage-sensitive and several ligand-gated currents typical for neuronal cells. The above HUCB-NSC characteristic conceivably implicates that cord blood-derived progenitors could be effectively differentiated into functional neuron-like cells in vitro.

Fabrication and characterization of protein arrays for stem cell patterning

Microarrays of fibronectin and other extracellular matrix (ECM) proteins were fabricated on plasma-deposited poly(ethyleneoxide) (PEO-like) film coated glass slides to study adhesion of stem cells. The arrays were generated by using a non-contact printing technology. The stability and the quality of the spots of fibronectin, used as protein model, were assessed by time of flight secondary ion mass spectrometry (ToF-SIMS), ellipsometry and atomic force microscopy (AFM). It was found that saturation with a mass density of 112 ± 4 ng/cm2 is reached when protein solutions at concentrations higher than 84 µg/ml are spotted. Fibronectin on the surface form a uniform sub-monolayer with a surface coverage that depends on the spotting solution concentration, as qualitatively demonstrated by AFM measurements. The active conformation of the spotted fibronectin was verified by performing an immunoassay with antibodies specific for the fibronectin RGD sequence by surface plasmon resonance (SPR) imaging. An immunorecognition efficiency of up to 22% was found for a spot with 3% coverage as estimated by ellipsometry. Human umbilical cord blood neural stem cells (HUCB-NSCs) were cultured on different ECM proteins (fibronectin, laminin, collagen I, collagen III and collagen V) arrays and showed protein concentration dependent adhesion on the micro-spots. The cell nuclei were stained for cell counting and preliminary specific cell staining was performed to evaluate the differentiation stage of HUCB-NSCs on such spots. The array platform developed in this study provides a promising approach to investigate in high throughput manner how surfaces patterned with extracellular matrix (ECM) proteins influence stem cell adhesion and development.

A novel, neural potential of non-hematopoietic human umbilical cord blood stem cells.

From the time of discovery that among the cord blood mononuclear cell population there are cells capable of changing their fate towards the neural lineage and producing functional neurons and macroglial cells, our attempts have been focused on the understanding of the underlying mechanism of this transition. We have deciphered the first steps of neural stem/progenitor gene induction in aggregating culture of cord blood mononuclear cells, their rapid phenotypic conversion under the influence of neuromorphogenic signals due to mitogen activation and their ability to expand and develop a prototypic, long-living line with neural stem cell properties. Evidence has accumulated that human umbilical cord-derived and neurally committed cells, due to their capacity for self-renewal, multilineage differentiation, plasticity and ability for long-lasting growth in vitro, provide unique material for the cell therapy of a wide spectrum of neurological diseases. The putative regenerating potential of these cord blood-derived neural stem/progenitor cells was evaluated after transplantation in experimental models of brain injury. In spite of initial promising data, the results indicate an urgent need to improve available animal model protocols in order to increase immuno-tolerance toward transplanted human cells.

Delayed induction of apoptosis by ammonia in C6 glioma cells.

Ammonia is a neurotoxin whose administration in large doses causes coma and death of the exposed animals, but whether and in what degree these whole body effects are related to the death of CNS cells is not known. Since the downstream effects of ammonia in cultured CNS cells appear to be partly mediated by overactivation of several putative signalling mechanisms characteristic for the apoptotic program, we speculated that ammonia neurotoxicity may be apoptogenic. In this study, C6 glioma cells grown in 2% serum were exposed to 5 mM or 10 mM NH(4)Cl (ammonia) for 96 h and tested for the appearance of apoptosis by (a) Hoechst staining, (b) TUNEL reaction and (c) DNA ladder, at different times of exposure. In cultures exposed to either 5 mM or 10 mM ammonia, about 10% of the cells were found to enter apoptosis at 48 h of exposure, and the number of apoptotic cells rose to 30% at 72 h, and to 50% at 96 h of exposure, respectively. The first transduction signal purportedly involved in apoptosis, activation of PKCalphabeta, was transient and appeared already after 3-6 h of treatment. Coincident with pronounced manifestation of apoptosis (at 72 h and even more at 96 h of exposure) was an increased transfer of the transcription factor NFkappaB from cytoplasmto nucleus as revealed by EMSA assay. The number of cells affected by ammonia-induced apoptosis was markedly reduced by incubation with a NOS inhibitor, L-NAME at 100 microM concentration. The results indicate that ammonia-induced apoptosis is a result of a complex interplay of at least three signalling molecules: NO, PKC and the transcription factor NFkappaB, with NFkappaB being possibly involved in the induction of iNOS and generation of toxic levels of NO in the cells.