Using Human-Induced Pluripotent Stem Cells (iPSC) …Using Human-Induced Pluripotent Stem Cells...
Transcript of Using Human-Induced Pluripotent Stem Cells (iPSC) …Using Human-Induced Pluripotent Stem Cells...
The developing brain is vulnerable to chemical-induced injury yet many chemicals remain
inadequately tested for developmental neurotoxicity (DNT). In an effort to develop and characterize
an in vitro model system for DNT screening, we exposed human iPSC-derived neurons to a diverse
set of 80 chemicals (e.g., neurotoxicants, drugs, pesticides, flame retardants (FRs), polycyclic
aromatic hydrocarbons (PAHs)) across a 6-point concentration range (~0.3 to 100 μM) in 384-well
plates. Using HCS imaging, effects on neurite outgrowth parameters (total outgrowth, processes,
branching) and cell viability were monitored after 72 h of exposure. Also, mitochondrial membrane
potential (MMP) was evaluated at 1 h to assess the potential contribution of MMP to altered neurite
outgrowth. The assay-specific noise threshold was calculated based on DMSO control variability and
concentration-response profiles were evaluated using a Hill model to derive benchmark
concentration (BMC) point-of-departure values. Following assay validation with controls and test
replicates, chemicals were ranked by toxicity and selectivity (i.e., effects on neurite outgrowth
parameters independent of cytotoxicity). Neurite total outgrowth and branching were the most
sensitive endpoints; 16 chemicals had an effect on neurite outgrowth independent of cytotoxicity.
The pesticide rotenone was the most selective, while1 FR (triphenyl phosphate) and 3 PAHs
(chrysene, dibenz(a,h)anthracene, acenaphthylene) inhibited neurite outgrowth. Of the 80
compounds, 39 decreased MMP and 9 were active in both assays, which might indicate that
alterations in MMP are linked with neurite outgrowth inhibition for these compounds. These studies
have important implications for moving the DNT field forward from a more traditional assessment in
animals to implementing novel and improved methodologies and should allow for a more rapid
screening and prioritization of potential neurotoxicants.
Abstract
Objective Results: BMC values
Results: Effects on neurite outgrowth &
mitochondrial function
This research was supported by the NIH, National Institute of Environmental Health
Sciences. Drs. Kevin Crofton, William Mundy, Stephanie Padilla, and Timothy Shafer at the
USEPA are acknowledged for their assistance with identifying the compounds to include in
the 80 compound library. We thank Dr. Brad Collins and Stephanie Holmgren (NTP, NIEHS)
for their assistance in assembling the 80 compound library and performing a detailed
literature search on the compounds, respectively.
Acknowledgments
References
Materials & Methods
Future Directions
Results: Assay Variability & Reproducibility
Using Human-Induced Pluripotent Stem Cells (iPSC) in High-Content Screening (HCS) to Prioritize
Chemicals for Developmental Neurotoxicity Testing Raymond Tice1, Fred Parham1, Jui-Hua Hsieh1, Mamta Behl1, Oksana Sirenko2, Evan Cromwell2 , Susan DeLaura3, Kristen Ryan1
1 NIH, NIEHS/DNTP, Research Triangle Park, North Carolina, United States; 2 Molecular Devices, Sunnyvale, California, United States; 3 Cellular Dynamics International, Madison, Wisconsin, United States
1. Characterize the neurite outgrowth assay as a suitable high throughput screen for a NTP
neuro-specific assay battery.
2. Explore the relationship between mitochondrial dysfunction and inhibition of neurite outgrowth.
3. Prioritize NTP chemicals of concern (e.g., FRs, PAHs) for more comprehensive in vivo hazard
characterization studies.
NT/DNT (38) NTP Nominations/Others (33)
1-methyl-4-phenylpyridinium iodide 2- Ethylhexyl diphenyl phosphate (EDHP)*
2-Methoxyethanol 2,2',4,4',5,5'-Hexabromodiphenyl ether*
3,3'-Iminodipropionitrile 2,2',4,4',5-Pentabromodiphenyl ether*
5-Fluorouracil 2,2'4,4'-Tetrabromodiphenyl ether*
6-Hydroxydopamine hydrochloride 3,3’,5,5’-Tetrabromobisphenol A*
6-Propyl-2-thiouracil Isodecyl diphenyl phosphate*
Acetic acid, manganese(2+) salt Phenol, isopropylated, phosphate (3:1)*
Acrylamide Tert-Butylphenyl diphenyl phosphate*
Aldicarb Tricresyl phosphate*
Bis(tributyltin)oxide Triphenyl phosphate*
Bisphenol A Tris(2-chloroethyl) phosphate*
Captan 4-H-Cyclopenta(d,e,f)phenanthrene†
Carbaryl Acenaphthene†
Chlorpyrifos (Dursban) Acenaphthylene†
Colchicine Anthracene†
Deltamethrin Benz(a)anthracene†
Di(2-ethylhexyl) phthalate Benzo(a)pyrene†
Diazepam Benzo(b)fluoranthene†
Dichlorodiphenyltrichloroethane (DDT) Benzo(e)pyrene†
Dieldrin Benzo(k)fluoranthene†
Diethylstilbestrol Benzo[g,h,i]perylene†
Heptachlor Chrysene†
Hexachlorophene Dibenz(a,h)anthracene†
Hydroxyurea Dibenz[a,c]anthracene†
Lindane Fluorene†
Methyl mercuric (II) chloride Naphthalene†
n-Hexane Phenanthrene†
Parathion Pyrene†
Permethrin 1-ethyl-3-methylimidazolium diethylphosphate¥
Phenobarbital sodium salt 2,3,7,8-Tetrachlorodibenzo-p-dioxin¥
Phenobarbitol Berberine chloride¥
Rotenone Carbamic acid, butyl-, 3-iodo-2-propynyl ester¥
Tebuconazole Manganese, tricarbonyl[(1,2,3,4,5-.eta.)-
Tetraethylthiuram disulfide 1-methyl-2,4-cyclopentadien-1-yl]-¥
Thalidomide Negative Controls (5)
Toluene Acetaminophen
Valinomycin Acetylsalicylic acid
Valproic acid sodium salt D-Glucitol
L-Ascorbic acid
Saccharin sodium salt hydrate
NT/DNT = compounds with reported neurotoxicity and developmental
neurotox1,2
Test replicates in italics were used to examine assay reproducibility
NTP Nominations = * Flame retardants (FRs) and † Polycyclic aromatic
hydrocarbons (PAHs) ¥ Others
iPSC-derived iCell® Neurons
Cells were received cryopreserved from Cellular
Dynamics International (CDI, Madison WI)
• Cells were thawed and plated according to CDI’s
protocol.
• iCell neuron methods and use in high-content toxicity
assays were previously evaluated by Sirenko et al.
(2014).
Neurite Outgrowth Assay
• Compounds were tested in duplicate at each
concentration (0.3 -100 μM) for 72 hr on laminin
coated 384–well plates with ~7500 cells/well.
• Cells were stained with Calcein AM & Hoescht dye
(Invitrogen, Carlsbad, CA) for 30 min prior to imaging
on an Image Xpress XL Instrument (Molecular
Devices, Sunnyvale, CA).
• Images were acquired using 10X or 20X objectives
and multiple exposures.
Mitochondrial Membrane Potential (MMP) Assay
• Compounds tested in duplicate at each concentration
(0.1-30 μM) for 1 hr on laminin coated 384–well plates
with ~7500 cells/well.
• Effects were monitored by the addition of the
mitochondria active dye JC-10 (ATT BioQuest Inc.,
Sunnyvale, CA) for 30 mins prior to imaging on an
Image Xpress XL Instrument (Molecular Devices,
Sunnyvale, CA).
• Images were acquired using 10X or 20X objectives
and multiple exposures.
Figure 1. Neurite Outgrowth Endpoints
Viable Cells: Number of viable cell bodies in the
image
Total Outgrowth: Total length of skeletonized
outgrowth in mm (corrected for diagonal lengths)
Total Processes: Number of outgrowths in the
image that are connected to cell bodies
Total Branches: Total number of branching
junctions in the image
Cell Body
Processes (4)
connected to
cell body Branch
Benchmark Concentration (BMC) Analysis
Dose-response information is critical for ranking
chemicals for toxicity or safety. However, robust
calculation of half-maximum activity responses
(i.e., AC50) is limited by the need for two
asymptotes. High concentration response
plateaus are often not present, leading to a need
for an improved analysis method. To overcome
this limitation, we applied the BMC analysis
method developed by the U.S. Environmental
Protection Agency for analysis of toxicity test
data3,4 to the multi-parametric neurite outgrowth
assay. Response data were normalized by
dividing by the mean of the dimethyl sulfoxide
(DMSO) control wells. Concentration-response
was modeled using a Hill model. The model
parameters were optimized by least-squares
fitting using MATLAB.
Model equation: f(x)=v0 + (vmax-v0)* xn/(kn + xn);
v0 is fixed at 1 (mean value of DMSO control data)
Figure 2. (A) Box plot showing the variance within DMSO controls per endpoint for 3 standard deviations (SDs). (B) 3SD values were used as a the benchmark response [BMR] (i.e., noise threshold) for data modelling and
benchmark concentration (BMC) calculations. (C) Concentration response curves and BMCs (μM) calculated for duplicates within the 80 compound library. For each plot, data points are represented by an asterisk (*) for
each concentration [log10 scale] for each endpoint. The Hill model was used to fit the data and estimate the BMC [Filled Circles: Red = Total Outgrowth; Green = Total Processes; Blue = Total Branches; Black = Viable cells].
A C
Ray I am putt
Results: Compound effects on mitochondrial membrane potential
Selective Compounds in the
Neurite Outgrowth Assaya
Category
Neurite
Outgrowth
BMC (μM)
MMP
BMC (μM)
Rotenone NT/DNT 1.37 1.60
Dieldrin NT/DNT 4.91 0.88
Hexachlorophene NT/DNT 8.12 0.06
Dibenz(a,h)anthracene PAH 7.91 30.00
Diethylstilbestrol NT/DNT 20.84 3.97
Aldicarb NT/DNT 21.14 30.00
Chrysene PAH 10.11 30.00
Carbaryl NT/DNT 21.65 1.29
Diazepam NT/DNT 21.82 30.00
Acenaphthylene PAH 24.14 12.59
Di(2-ethylhexyl) phthalate NT/DNT 25.06 30.00
Triphenyl phosphate FR 13.33 6.24
Methyl mercuric (II) chloride NT/DNT 0.55 1.86 Manganese, tricarbonyl[(1,2,3,4,5-.eta.)-1-
methyl-2,4-cyclopentadien-1-yl]- Other 28.52 30.00
Tebuconazole NT/DNT 29.14 30.00
Tetraethylthiuram disulfide NT/DNT 10.29 6.61
a compounds listed in order of selectivity scores (SS) (high to low) for neurite outgrowth by the most sensitive
endpoint
Gray colored box: compounds affected both neurite outgrowth and MMP < 30 μM
NT/DNT = neurotoxicant/developmental neurotoxicant
FR = flame retardant; PAH = polycyclic aromatic phosphate; BMC = benchmark concentration value
Compound BMC (µM)
Valinomycin 0.04
Hexachlorophene 0.06
1-ethyl-3-methylimidazolium diethylphosphate 0.51
Triphenyl phosphate 0.76
Dieldrin 0.88
Carbaryl 1.29
Rotenone 1.60
Methyl mercuric (II) chloride 1.86
Phenol, isopropylated, phosphate (3:1) 2.65
Carbamic acid, butyl-, 3-iodo-2-propynyl ester 2.81
A
B
C
1. Mundy et al. (2009) The Toxicologist
2. Crofton et al. (2011) ALTEX
3. US EPA (2012) Benchmark Dose Technical Guidance
http://www.epa.gov/raf/publications/pdfs/benchmark_dose_guidance.pdf
4. Robin X et al. (2011) BMC Bioinformatics 12: 77
• Automated high throughput, high content imaging can be utilized to evaluate the
effects of chemicals on neurite outgrowth in human iPSCs.
• Based on the variability in the solvent control wells, an assay specific SD was
calculated and 3 SD was used as a conservative measure of an acceptable
benchmark response (BMR) for BMC analysis.
• The endpoint with the least noise in the solvent control data was viable cells
(measure of cytotoxicity), followed by neurite total outgrowth, total processes, and
total branches.
• The majority of actives in this assay were identified by effects on total branching >
total outgrowth > total processes.
• Duplicate chemicals show similar concentration responses & calculated BMC values
indicating good internal assay reproducibility.
• 9/76 unique compounds inhibited both MMP (1 hr) and neurite outgrowth (72 hr) and
belong to diverse structural classes such as pesticides, drugs, metal-containing
compounds, FRs, and PAHs, suggesting that mitochondrial dysfunction may be an
upstream signaling event of neurite outgrowth inhibition.
• Compare the results from this assay with data generated by collaborators and in the
literature.
• In vitro actives in neurite outgrowth assay
• Known in vivo neurotoxicants and developmental neurotoxicants
• Prioritize suspected neurotoxicants or chemical classes of NTP interest (i.e., FRs,
PAHs) for in vivo hazard characterization & targeted/mechanistic follow-up studies.
V(0)
AC50 (half-
maximal activity
concentration)
BMC
Vmax Hill Model
BMR for
each
endpoint
Accurate
estimation
depends on high
quality data curve
Resp
on
se
Dose
B
Total
Outgrowth
Total
Processes
Total
Branches
Viable
Cells
Saccharin sodium salt-1 Saccharin sodium salt-2 Methyl mercuric (II) chloride-1 Methyl mercuric (II) chloride-2
Deltamethrin-1 Deltamethrin-2 Triphenyl phosphate-1 Triphenyl phosphate-2
Re
sp
on
se
3SD=0.09 3SD=0.21 3SD=0.20 3SD=0.11
Figure 3: Stem plot showing a ranking of the 80 compounds by lowest BMC per endpoint.
For all compounds with a BMC value < 100 μM, a comparison is shown between the BMC for the
most sensitive endpoint and the BMC for viability as a marker of cytotoxicity. For several
compounds, the BMC was not estimated because no effect was observed over the concentration
range tested (0.3 – 100 μM). BMC = benchmark concentration.
BMC (μM)
Ranking of all 80 Compounds: Lowest BMC
Total Outgrowth
Total Processes
Total Branches
Viable Cells
KEY
Total Outgrowth
Total Processes
Total Branches
Viable Cells
KEY
• 41/80 (51%) of the compounds had a effect on at least 1 endpoint in
the neurite outgrowth assay
BMC μM
Results: Compound-specific effects
on neurite outgrowth
Control 0.3 μM Rotenone 3 μM Rotenone
Figure 5. High content imaging and dose
response modeling for rotenone. (Left)
Modelled concentration response curves
from duplicate data points (*) and BMCs for
each neurite endpoint demonstrate that
effects on neurites occur at lower
concentrations than effects on cell viability.
BMC = benchmark concentration and is
represented by a symbol (circle or triangle).
(Below) Neuronal bodies and neurites were
visualized (20X) with Calcein AM dye (green)
following a 72 hr exposure using iPSC-
neurons.
Figure 4: Stem plot showing 16 compounds which were identified as selective for inhibition
of neurite outgrowth. Selectivity is the extent to which the BMC for the most sensitive neurite
endpoint is less than the BMC for cytotoxicity. Selectivity was calculated for compounds with BMCs
< 100 μM by subtracting the log10 BMC for each endpoint from the log10 BMC value for viable cells
(marker of cytotoxicity). BMC values of > 100 µM for viable cells are treated as 100 µM. This will
underestimate selectivity. Selectivity calculations of 0.5 (i.e., the log10 difference between two
consecutive concentrations) was required for a compound to be deemed selective. BMC =
benchmark concentration.
16 compounds show selective inhibition of neurite outgrowth
Rotenone
Table 1: 80 compound library
Table 2: Top 10 inhibitors of MMP
Table 3. Evaluating the contribution of mitochondrial function on neurite outgrowth
• 39/80 (49%) of the compounds had a effect on mitochondrial membrane potential (MMP)
BMC (μM)
Disclaimer: This data does not reflect the final opinion of the National Toxicology Program
Conclusions
Figure 6. Heatmap of BMC values for all compounds tested in the 1 hr MMP assay. Blue = decreasing responses in MMP or cell
viability. White = no effect in the concentration range tested (0.1 – 30 μM).
30
μM
0
10
0.1
0.1
5