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Determination of Toxicity Effects on Maturing Human Neuronal Cells

Characterization of Neural DifferentiationIn Vitro

Summary

Funding

Introduction

• EPA-G2012-STAR-F1 Human Neural Stem CellMetabolomic, Cellular and Organ Level AdverseOutcome Pathway Relationships For EndocrineActive Compounds

• University of Georgia Interdisciplinary ToxicologyProgram partial support for Xian Wu

• BPA did not inhibit HuC/D+ neural cell differentiationor survival in WOS1 using the doses tested.

• In WOS2, the highest dose of BPA (10 μM) decreasedthe number of HuC/D+ cells compared to non treatedcells.

• BPA decreased branch point per neuron starting at 10μM in WOS1 and 0.1 μM in WOS2

Multiple central nervous system disordersmay be due to extended toxicant exposureduring neural development windows ofsusceptibility (WOS) continuums. Humanstem cell can likely model differentdevelopmental WOS

AimsCharacterize bisphenol-A (BPA) effects onneural differentiation and cell function(neurite outgrowth) using differentiatinghuman pluripotent stem cell derived neuralprogenitor (hNP1; ArunA Biomedical) duringtwo WOS.WOS1: undifferentiated hNP1 to neural cellsexpressing HuC/D (Fig 3)WOS2: HuC/D to homogenous MAP2+extending neurites (Fig 3)

Bisphenol A Effects on In Vitro Human Neural Development Was Window of Susceptibility Dependent

Xian Wu1,2, Anirban Majumder3, Steven L. Stice1,2,3

1 Interdisciplinary Toxicology Program, 2 Regenerative Bioscience Center, The University of Georgia, 3 ArunA Biomedical, Inc. Athens, GA, USA

A B

MAP2 / Hoechst

C

D E GF

D E F

A B C

HuC/D / Hoechst

G

Materials and Methods

Cell culture: Human neural progenitor (hNP1)cells were seeded in a 96 well plate indifferentiation media at 15,000 cells per wellfor 2 weeks using ArunA neural media andsupplement. Cells were maintained in ahumidified incubator at 37°C with a 95%air/5% CO2 atmosphere.Chemical treatment: 0.01, 0.1, 1, 10 μMBisphenol A applied to cells 6 h after platingand maintained for whole WOS (14 DIV)Immunocytochemistry: Primary antibodiesapplied as follows: HuC/D 1:40, A21271(Invitrogen Corp., Carlsbad, CA), MAP2(microtubule-associated protein 2) 1:400,AB5622 (Chemicon, Temecula, CA) and βIII-tubulin 1:300, AB18207 (ABCAM, Cambridge,MA). Cells were then incubated with a 1:400dilution of DyLight® 594-conjugated donkeyanti-mouse IgG or DyLight® 488-conjugateddonkey anti-rabbit IgG secondary antibody.Cells were then incubated in 0.1%Hoechst33342 dye in high salt buffer for 20 min.Measurements of differentiated neuron cellmorphology: Stained cell cultures in 96 wellplates were loaded into a Cellomics ArrayScanVTI HCS reader high-content imaging system(ThermoFisher Scientific, Waltham, MA) forautomated image acquisition andmorphometric analyses. Image acquisition andstorage was performed using the vHCS Scansoftware package and image analysis wasperformed in real-time with a manuallyoptimized version of the Cellomics NeuralProfiling Bioapplication and Cellomics TargetActivation Bioapplication.

Longitudinal HuC/D Expression

Automated Measurement of MAP2 Expression and Neurite Outgrowth

DIV 0 DIV14 DIV 28

0-14 DIV Exposure(WOS 1) 14-28 DIV Exposure (WOS 2)

Early Neuron Later NeuronNeural Progenitor

A B

Hu C/D

MAP2

Neural Progenitor (DIV 0) Neuron (DIV 28)

Reference

Fig. 1 . Automated measurement of HuC/D expression during differentiation. HuC/D expresses only in post mitoticneurons. A ,D: nuclear identification B,E: HuC/D staining and identification C,F: Pseudocolored composite image.; bluetrace = nuclei, red trace = HuC/D positive neuron, yellow trace=rejected nuclei. Red dotted boxes in A-C denote areasmagnified to illustrate image tracing in panel D-F. G: Data are presented as mean ± S.D. Each bar represents n = 6replicates. The asterisk (*) indicates significant difference from undifferentiated neural progenitor cells and Δindicates significant difference between groups(P < 0.05). Scale bars = 50 µm.

Fig. 2 . Differentiating hNP cultures were fixed at different time points for analysis following immunocytochemistrystaining with MAP2 antibody. A (channel 1): Nuclei stained with Hoechst 33342, live cell nuclei (blue trace). B (channel2) Cell body masks and neurite trace based on MAP2 expression; Blue trace = accepted cell, Red trace = rejected cell,Purple line = neurite. C. Pseudocolored composite image combining channels 1 and 2. D. MAP2 positive cell numberwas quantified at different days. E. neurite total length per neuron; F. neurite total count per neuron; G. branchpointtotal count per neuron. Data is presented as mean± S.D. n = 6 replicates. (*) indicates significant difference betweengroups (P < 0.05). Scale bars = 50 µm.

BPA WOS 1: no change in maturation or cytotoxicity with high dose decreased neurite outgrowth

BPA WOS 2: high dose increased cytotoxicity and decreased branching at lower dose

G

Fig. 3 . Human neural progenitor (hNP1) cells (A) were differentiated in vitro to neurons (B). Two test phases were established totest the effects of compounds on the continuum of differentiation and maturation in vitro. Scale bars = 100 µm.

A B C D

A B C D

Harrill, J.A., et al., Quantitative assessment of neuriteoutgrowth in human embryonic stem cell-derivedhN2 cells using automated high-content imageanalysis. Neurotoxicology, 2010. 31(3): p. 277-90.

Conclusion

Fig. 4 . Quantification of neurons (HuC/D+). Human neural progenitor cells were cultured on 96 well plates and continuouslyexposed to a range of doses of BPA for 2 weeks. A) Neuron density was measured as an indicator of cell health. B,C,D) Averagenumber of neurites per neuron, total neurite length per neuron and total branch points per neuron were also measured. All dataare presented as % from untreated control wells. (#) indicates significant difference from control group (P < 0.05)

Fig. 5 . Quantification of neurons (HuC/D+). Early neuron cells were cultured on 96 well plates and continuously exposed to arange of doses of BPA for 2 weeks. A) Neuron density was measured as an indicator of cell health. B,C,D) Average number ofneurites per neuron, total neurite length per neuron and total branch points per neuron were also measured. All data arepresented as % from untreated control wells. (*) indicates significant difference from control group (P < 0.05)

• hNP1 cells are the in vitro equivalent of developingneuroepithelial cells in the human neural tube.Here, results suggested that BPA has little effect onin vitro equivalent neural tube stage and earlyneural differentiation (WOS1).

• More fully differentiated neural stages (WOS2) weresensitive to increasing doses of BPA, specifically theability of neurites to generate or maintain branchpoints was diminished, an indication of decreaseddendritic arborization.

• Levels of neural arborization is associated withseveral neural disorders and this is the first humanin vitro assay to suggest that BPA preferentiallyaffected branching of more fully differentiatedneurons.