optimization of neurosphere assays using human neuronal...

Columbia International Publishing American Journal of Tissue Engineering and Stem Cell (2015) Vol. 2 No. 1 pp. 7-18

Research Article

______________________________________________________________________________________________________________________________ *Corresponding e-mail: [email protected] 1 Center for Environmental Risk Research, National Institute for Environmental Studies, 16-2 Onogawa,

Tsukuba 305-8506, Japan 2 Department of Life and Environmental Science, Major of Bioindustrial Sciences, The University of Tsukuba,

1-1-1 Tennodai, Tsukuba, Ibaraki 305-0006, Japan 3 Laboratory of Offshore Medical Physiology, Department of Medical Physiology, Hainan Medical College, No.

3, College Road, Longhua District, Haikou City, Hainan Province, China

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Optimization of Neurosphere Assays using Human Neuronal Progenitor Cells for Developmental

Neurotoxicity Testing

Yang Zeng1,2, Yang Wang3, Qin Zeng1 , Hiroko Nansai1, Zhenya Zhang2, and Hideko Sone1*

Received 20 August 2015; Published online 17 October 2015 © The author(s) 2015. Published with open access at www.uscip.us

Abstract The presence of neurotoxicants in food and the environment has been implicated in increases in neuronal disease in advanced countries. To use neurospheres formed from human neuronal progenitor cells (hNPCs) for risk evaluation of chemical exposure, the neurospheres need to be produced under stable and efficient conditions. We have developed a novel culture system to achieve this goal. We cultured spheres of hNPCs on a laminin 511 (LN511) matrix to elucidate whether LN511-enriched basement membrane promotes neurite outgrowth from hNPCs. Cells cultured on the LN511 matrix exhibited increased neurite lengths compared with those cultured on laminin 111 (LN111). The results showed that LN511 is an appropriate and stable matrix for neurosphere culturing, and that integration of lamina-dense structure provides an ideal surface for neurite outgrowth. These extracellular microenvironments are believed to provide binding sites for neural progenitor cells, activating intracellular signaling pathways and promoting sphere formation and neurite differentiation. Benzo[a]pyrene and 5-Azacytidine were applied to our established system. Both chemicals significantly inhibited cell migration and induced apoptosis. It is concluded that our novel culture system can efficiently and stably be established and is a good tool for investigations of the roles of neurotoxins or therapeutic agents in neuronal disease. Keywords: Embryonic formation; Laminin 511; Neurite outgrowth

1. Introduction Accumulating evidence indicates that many environmental toxicants are linked to neuronal disease, and some environmental toxicants are involved in developmental neurotoxicity (DNT) in humans

Yang Zeng, Yang Wang, Qin Zeng, Hiroko Nansai, Zhenya Zhang, and Hideko Sone / American Journal of Tissue Engineering and Stem Cell (2015) Vol. 2 No. 1 pp. 7-18

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and rodents (Harry et al., 2014; Myers et al., 2015). It is well known that in vivo assays give more information about what is really happening inside organisms, while estimates based on two-dimensional in vitro observations might be very different from the actual in vivo situation. However, toxicity testing of chemicals in in vivo experimental studies has led to serious ethical concerns. Moreover, in vivo assays are too expensive to acquire batch dose data. Therefore, the development of alternative procedures and models is necessary. A three-dimensional culture system has the advantages of being convenient, efficient and similar to the in vivo situation, with improved intercellular interactions, and the model is able to replicate basic processes of fetal brain development such as proliferation, differentiation and apoptosis (A. Orza et al., 2011; A. I. Orza et al., 2014). The use of human neurospheres, which are a kind of three-dimensional culture system likely to imitate the basic processes of brain development, has been a promising approach for DNT testing (Moors et al., 2009). The developing nervous system is highly vulnerable to the adverse effects of chemical agents. Therefore, we developed a neurosphere culture system based on human neural progenitor cells (hNPCs) and exposed the neurospheres to chemicals during neuronal development to detect DNT. The basement membrane (BM) is a key component of a neurosphere testing model. The three-dimensional system basement membrane provides a scaffold for embryonic stem cell differentiation and is known to regulate adhesion, migration, proliferation and differentiation (Halper et al., 2014). Poly-L-ornithine (PLO), a highly positively charged amino acid chain, is generally used as a coating reagent to promote cell adhesion in culture. PLO alone or in combination with Fibronectin or laminin-I is frequently used to enhance attachment and differentiation of various types of neurons and neural stem cells (Choy et al., 2000; Darrabie et al., 2005). laminins synthesized membrane surfaces has a highly integrated structure composed of extracellular matrix (ECM) molecules, develop niche microenvironment inducing an activation of transcellular membrane signals transmission in embryonic stem cells differentiation. laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed alpha, beta and gamma (Ido et al., 2007). In this study, we attempted to acquire the efficient progenitor cells derived neurite outgrow on a substrate that integrates several laminin subunits including laminin 511 (LN511) and laminin 111 (LN111). LN511 is the most widely expressed laminin in the body (El-Ghannam et al., 1998). The human ES cell membrane has high affinity for intact LN511, which is effective for maintaining pluripotency, supporting efficient adhesion and expansion of dissociated human pluripotent stem cells (Kikkawa et al., 2007). LN111 is present in the early embryo and later in certain epithelial cells, but it is a rare isoform in vivo (Bougas et al., 2014; Ekblom et al., 2003). Neurite differentiation protocols enable the creation of different subtypes of neurons to study fate decision pathways (Gaspard et al., 2010), and have further utility as assay systems to detect adverse effects of toxicity in neuronal development.

The novel culture system was exposed to two chemicals to find out whether it can be used to evaluate developmental neurotoxicity. Benzo[a]pyrene (BaP), a well known carcinogen, is widespread in the environment. Although the neurotoxic effect of BaP has not drawn much attention, adverse effects of BaP on learning and memory have been reported (Liang et al., 2014). Neuronal apoptosis plays a major role in BaP-induced neurotoxicity (Nie et al., 2014). 5-Azacytidine (5AZ) is a potent inhibitor of DNA methylation (Hossain et al., 1997a). It has been reported that 5AZ


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induces toxicity in PC12 cells and in rat brain, however, no neurotoxicity has been found (Hossain et al., 1997b; Ueno et al., 2002b).

The aim of this study was to determine an optimal basement membrane culture system and establish a neurosphere model to detect developmental neurotoxicity. The results revealed that a LN111 coating system cannot support the neurospheres for long enough. A POLY-L-ornithine /LN511+111 coating system showed the best attachment, however, the neurospheres that formed were too extended to make a stable system. The LN511 and LN511+111 coating systems showed similar performance, but the LN511 coating system is more convenient and efficient. Therefore, it is suggested that LN511 single coating is best for establishing a stable and efficient culture system. Both BaP and 5AZ inhibited cell migration. BaP, but not 5AZ, inhibited neuronal differentiation. It is suggested that our neurosphere model can be used to evaluate developmental neurotoxicity.

2. Materials and Methods

2.1 Chemicals L-glutamine, Poly-L-ornithine and laminin from engelbreth-holm-swarm (laminin 111) were provided by Sigma-Aldrich (Tokyo, Japan). Human recombinant laminin (laminin 511) was obtained from BioLamina AB (Stockholm, Sweden). Benzo(a)pyrene and 5-Azacytidine was bought from Schell Chemical (Co, New York, N. Y). 2.2 hNPC sphere formation and neural cell differentiation on the extracellular matrix The human neuronal progenitor cells (hNPCs), derived from H9 human embryonic stem cells, were obtained from Chemicon-Millipore (ENStem Human Neural Progenitor Expansion Kit; Norcross, GA). For hNPC proliferation prior to the neural progenitor sphere (NPS) formation, preparations were incubated in poly-L-ornithine/LN111-coated 60-mm Petri dishes with NEM medium (ENStem-A Neural Expansion Medium(EMD Millipore, Japan) with Glutamine and FGF-2 supplement solution) and then hNPCs were seeded at a density of 2 × 106 cells/dish and incubated for 4 days. Preparations were then dissociated with accutase dissociation solution (ICT, FUAT104). NPS formation was conducted in Nunc low cell binding 96-well plates (Thermo Fisher Scientific Inc., Waltham, MA) with a density of 6 × 103 cells/well in NEM medium for 5 days. In the final step, NPS were transferred and seeded to 48-well plates in which the wells were coated with LN511 (BioLamina AB), a mixture of LN511 and LN111, or mixture of poly-L-ornithine/LN111 and LN511, or poly-L-ornithine/LN111, separately. The wells contained Neurobasal (Gibco,Japan) medium containing 5 units/ml penicillin with 5 μg/ml streptomycin and Gibco supplement B27 (1/50), Glutamax-1(1/100, Gibco), N2 (1/100, WAKO,Japan) and BDNF (1/1000, Gibco). The spheres were continuously cultured for at least 5 days to induce mature neural cells. 2.3 Immunohistochemistry staining Neurospheres were fixed with 4% paraformaldehyde for 15 min at RT, and permeabilized by incubation for 30 min in 0.1% Triton X-100 (SigmaAldrich, Japan)PBS. After blocking non-specific

http://www.biocompare.com/16913-Human-Neural-Stem-Cell-Media/344709-ENStemAtrade-Neural-Expansion-Medium/


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binding with 0.1% bovine serum albumin (BSA) in PBS, the fixed neurospheres were incubated with primary antibodies (MAP2 monoclonal, SigmaAldrich, Japan) overnight at 4°C, and then for 1 h at 37 °C with goat anti-mouse IgG antibodies conjugated to Alexa Fluor 568 (orange fluorescent dye, Invitrogen, Life Technologies, USA) or Alexa Fluor 488 (green fluorescent dye, Invitrogen, Life Technologies). The neurospheres were then incubated with Hoechst 33342 work solution (Molecular Probes, Life Technologies) for 15 min at RT. The staining was visualized with a fluorescent microscope (Olympus IX70, Olympus Corporation, Tokyo, Japan).

2.4 Image analysis for neurite outgrowth Sphere area and neurite migration were investigated. Images for each neurosphere were acquired with a 10 x lens, and area of the core and sphere were measured using Image J. Corona areas were calculated (sphere area minus core area). For determination of neurite length/cell, the IN Cell Analyzer 1000 (GE Healthcare, Japan) was used to automatically acquire fluorescent images. 20 images obtained with a 10 x lens were used to analyze neurite length. Cell images were analyzed using IN Cell Developer box (GE Healthcare). Cellular nuclei were first segmented and counted using the Hoechst channel. The neuron cell bodies were then segmented and their associated neurites were identified using the MAP2 channel. Neurite length was identified for each cell body and neurite length/cell was calculated.

3. Results 3.1 Neurosphere formation and differentiation

To develop a neurosphere differentiation culture system, we utilized a three-dimensional embryoid aggregate protocol to examine neurosphere formation and differentiation using various types of coating matrices. Fig. 1A is the timeline schematic for this protocol, in which neuronal progenitor sphere (NPS) were formed by Day 5 and neural maturity was induced by Day 10. Neurospheres and expanding single layer hNPC coronas were formed on all types of membrane-coated surfaces by Day 8. The expanded corona area of neurosphere was observed in PLO/LN111 coated surfaces, but the neurosphere central area was weak and not well attached on coating surfaces at Day 10 (Fig. 1B). Comparing the three other types of coating membranes at Day 10, significant neurosphere expanding corona areas were observed in the PLO/LN511+111 coated surface (Fig. 1B). The neurosphere expanding corona areas were significantly increased in PLO/LN511+111 surface on Day 10 compared with other coating systems (Fig. 2). However, the corona areas were too expanded and not stable, suggesting that the PLO/LN511+111 surface is not suitable for neurotoxicity testing. For further investigation of the expanding corona region, the neurite length/cell were analyzed, however, no significant differences were found for neurite length/cell (Fig. 3B).


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Fig. 1. A, Schematic of sphere formation, differentiation and migration. hNPC were formed in 96-well plates with round bottoms and transferred to matrix-coated 48-well plates for matrix testing. B, images of neurospheres cultured with different matrices on Day 8 and Day 10.

Fig. 2. Quantitative analysis of expanding corona area (sphere area minus core area) in

neurospheres, examined by fluorescence microscopy. Data are expressed as mean ± SD for three independent experiments. *P < 0.05 was considered to be statistically significant differences between groups using ANOVA followed by the Tukey’s test.


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Fig. 3. Immunofluorescence of MAP2-positive hNPCs at expanding corona on LN511-coated plates,

LN511/LN111-coated plates and PLO - LN511/LN111-coated plates. Cells were stained with hoechst33342 (blue) and MAP2 (Alexa 488, green). A, Typical outgrowth of neurosphere. B, Quantitative analysis of neurite length / MAP2-positive cell and nuclei member counting. Data are expressed as mean ± SD for three independent experiments. Significant differences between groups using ANOVA followed by the Tukey’s test.


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3.2 Benzo(a)pyrene and 5-Azacytidine exposure To investigate whether the LN511 coating is appropriate for assessment of neurotoxicity of chemicals, neurospheres on LN511-coated surface were exposed to benzo[a]pyrene (BaP) and 5-Azacytidine (5AZ) from Day 2 to Day 5 (Fig. 4A). BaP at a concentration of 10−5 M significantly inhibited expanding corona areas (Fig. 4C). However, BaP did not affect neurite length/cell at any test concentrations (Fig. 4D). 5AZ at a concentration of 3x10−7 M significantly inhibited expanding corona areas, while no significant differences from controls were found for lower concentrations (Fig. 5B). However, no significant changes were found for neurite length/cell at test concentrations (Fig. 5C).


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Fig. 4. A, Schematic of chemical exposure of neurospheres. hNPC were incubated with chemicals

from Day 2 to Day 5 in 96-well plates with round bottoms. Cells were stained with hoechst33342 (blue) and MAP2 (Alexa 568, red). B, Typical outgrowth of neurosphere exposed to BaP. C, Quantitative analysis for corona area. D, Quantitative analysis for neurite

length/MAP2-positive cell and nuclei member counting. Data are expressed as the percentage of the control ± SD for three independent experiments. *P < 0.05 was considered to be statistically significant differences between the treatment group and the vehicle control using ANOVA followed by the Dunnett's post hoc test.


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Fig. 5. A, Typical outgrowth of neurosphere exposed to 5AZ. Cells were stained with MAP2 (Alexa

568, red). B, Quantitative analysis for corona area. C, Quantitative analysis for neurite

length/ MAP2-positive cell and nuclei member counting. Data are expressed as the percentage of the control ± SD for three independent experiments. ***P < 0.001 were considered to be statistically significant differences between the treatment group and the vehicle control using ANOVA followed by the Dunnett's post hoc test.

4. Discussion We developed a novel, efficient three-dimensional culture system for neurosphere formation and neuronal differentiation with a combination of neurosphere formation on U-bottom plates and differentiation on the extracellular matrix. LN511 strongly supported neurosphere culture for long periods. hNPCs showed the ability to differentiate to neuronal cells and neurites were well developed on a LN511-coated surface. This novel neurosphere culture system can be used to analyze neurotoxicity. Neuron migration can be analyzed by measurement of expanding corona areas. Neurite differentiation can be quantified by measuring neurite length/cell. We tested several kinds of coating system. The neurosphere expanding corona areas were significantly increased on PLO/LN511+111 surface on Day 10. The extended neuron was too much compare with other coating system. Moreover, the standard deviation value was high, suggesting that the PLO/LN511+111 surface is not as stable as the LN511 and is not suitable for neurotoxicity testing. The results suggest that neurospheres can be maintained and appropriately differentiated when cultured using a LN511 or LN511/LN111 coating system. The LN511-coated surface system is more efficient and easier to operate than LN511/LN111 coating system, therefore it is suggested that LN511 is the best coating system. Culture systems using LN511 have been reported previously (Miyazaki et al., 2012). 2D culture system of human embryonic cells were well established using LN511 coating surface. Basement membrane components play an important role in supporting the regional-specific differentiation of embryonic stem cells into hepatic endoderm. Using an HEK293 cell line (rLN10-293 cells) stably expressing laminin 511, embryonic stem cells could be induced to differentiate into definitive endoderm and subsequently into pancreatic lineages (Shiraki et al.,


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2011). Instead of using cell-generated laminin 511, we used a commercially produced laminin 511 to establish a culture system. We developed an efficient three-dimensional culture system for NS formation and neuron differentiation. This novel NS culture system was used to assess neurotoxicity in our previous study (Qin et al., 2012). It has been shown recently that Benzo[a]pyrene may induce neurotoxicity (Chepelev et al., 2015; Schellenberger et al., 2013). However, 5AZ can only induce acute toxicity in the nervous system via apoptosis (Hossain et al., 1997a; Ueno et al., 2002a; Weisman et al., 1985). Therefore, we used these two chemicals to evaluate our novel model to determine whether it can assess neurotoxicity. We found that both chemicals inhibit corona areas of neurospheres, suggesting that both chemicals may influence cell migration. Although there was no significantly changes of neurite length/cell for both chemicals, BaP induced decreases of neurite length/cell at concentrations of 10−5 M, 5AZ induced increases in neurite length/cell at a concentration of 3×10−7 M. It is suggested that BaP but not 5AZ induces neurotoxicity by inhibiting neurite differentiation. The results indicate that our novel culture system can be used to assess developmental neurotoxicity in human health. In conclusion, our study indicates that intact LN511 substrates are qualitatively important for efficient and stable hNPC differentiation. The addition of poly-L-ornithine is advantageous for hNPC proliferation, but not suitable for stable hNPC differentiation. Our established LN511 culture system can distinguish between neurotoxic and non-neurotoxic substances, and it is suggested that our novel model is a good tool for investigations of environmental neurotoxicity.

Acknowledgements We thank Dr. Zhi Bin Chen, Professor of Neurosciences, Hai Nan Medical College and Dr. Rendy Jensen, Huntsman Cancer Institute, University of Utah for advice in manuscript writing.

Funding Source This study was supported in part by the Grant in Aid for Scientific Research from the Ministry of the Health and Labor, and Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan (24241013) to H.S. Japan.

Conflict of Interest None

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