The Toxicologist - Society of Toxicology

and progression of neurological diseases. In conclusion, the potential human iPSC
approaches offer for translational toxicology and assessment of human risk factors
will be discussed.
2451 APPLICATIONS OF HUMAN STEM CELL
TECHNOLOGY TO NEUROTOXICOLOGY.
P. J. Lein. Veterinary Medicine, Molecular Biosciences, University of California Davis,
Davis, CA.
Brief opening remarks will introduce the audience to the topic of human pluripotent
stem cells and induced pluripotent stem cells (iPSCs) and provide an overview
of the technical challenges and exciting opportunities for neurotoxicology provided
by this evolving technology.
2452 USING NEURAL PROGENITOR CELLS IN HIGH-
THROUGHPUT SCREENS FOR DEVELOPMENTAL
NEUROTOXICANTS: TRIUMPHS AND TRAGEDIES.
T. J. Shafer, M. Culbreth, J. M. Breier and W. R. Mundy. Integrated Systems
Toxicology, U.S. EPA, Research Triangle Park, NC.
Current protocols for developmental neurotoxicity testing are insufficient to test
thousands of commercial chemicals. Thus, development of high-throughput
screens (HTS) to detect and prioritize chemicals that may cause developmental
neurotoxicity is needed to improve protection of public health. While a variety of
cell culture models are amenable to this approach, neuroprogenitor (NP) cells, particularly
those of human origin, may provide highly relevant models for developmental
neurotoxicity testing. Over the past 5 years, we have examined the utility of
different NP cell models for HTS assays of proliferation, apoptosis, viability and
differentiation. Using ReNcell CX cells, a human cortical NP cell, we measured
changes in proliferation (BrdU incorporation) induced by known antiproliferative
compounds as well as compounds (0.001-100 μM) known to cause developmental
neurotoxicity; non-neurotoxic compounds were without effect. Furthermore, we
demonstrated that this approach is amenable to HTS by screening a set of 309
chemicals for effects on proliferation. More recently, we demonstrated that ReNcell
CX cells are also useful for HTS assays for apoptosis (activated p53 and Caspase) by
identifying appropriate assay positive controls. We have conducted more limited assessment
of proliferation in other human NP cells. Recently, we compared ReNcell
CX cells to mouse cortical NP cells and demonstrated that both cell types are sensitive
to compounds known to alter proliferation and/or apoptosis. Development of
HTS assays for differentiation has been more challenging. NP cells tested to date
have not differentiated rapidly, and it has been difficult to identify a cellular marker
for committed neurons suitable for this endpoint. Our work demonstrates that NP
cells can be utilized in HTS assays for the important neurodevelopmental endpoints
of proliferation and apoptosis, but more development will be needed to use
these cells in assays for neuroprogenitor differentiation. (This abstract does not reflect
Agency Policy)
2453 TRENDS OF THE IPSC TECHNOLOGY FOR
BIOMEDICAL RESEARCH AND CELL THERAPY:
POTENTIAL OF PROTEIN-INDUCED IPSCS.
K. Kim. McLean Hospital/Harvard Medical School, Belmont, MA. Sponsor: A.
Bowman.
Widely established methods to generate induced pluripotent stem cells (iPSCs) require
the use of viral and/or genetic materials that likely integrate into the chromosomal
DNAs with unknown genetic changes. The Kim lab has developed a method
to generate stable iPSCs from human fibroblasts by directly delivering four reprogramming
proteins (Oct4, Sox2, Klf4, and c-Myc) fused with a cell-penetrating
peptide (CPP). Approaches such as these have the potential to decrease confounding
variability in iPSC outcome measures due to the use of viral and chemical mediators
of reprogramming that have the potential to permanently influence gene expression,
neuronal differentiation and toxicant sensitivities. In support of this, we
found that precursor cells derived from lentivirus- and retrovirus-based human
iPSCs exhibit early senescence and significant apoptotic cell death whereas those
from protein-based human iPSCs and human ESCs do not show such abnormal
phenotypes. This presentation will discuss our comparative studies of neural differentiation
and their cellular and molecular characteristics using human iPSC lines
526 SOT 2011 ANNUAL MEETING
that are generated by different methods. These studies will enable the design of the
robust methodology and quality control of individual iPSC lines that is necessary
for eventual high throughput analysis of gene-environment interactions across patient-derived
iPSC lines.
2454 IN VITRO MODELS OF ANGELMAN AND PRADER-
WILLI SYNDROME VIA INDUCED PLURIPOTENT
STEM CELL TECHNOLOGY.
M. Lalande. Genetics and Developmental Biology, University of Connecticut Health
Center, Farmington, CT.
The recent discovery of nuclear reprogramming of human somatic cells into induced
pluripotent stem (iPS) cells offers an innovative and relevant approach to
study human genetic and neurogenetic diseases such as Angelman syndrome (AS)
and Prader-Willi syndrome (PWS). We have reprogrammed AS and PWS patient
dermal fibroblasts using the four Yamanaka factors (POU5F1 [OCT4], SOX2,
MYC and KLF4) and LIN28 delivered to the cells with retroviral vectors. Using
this approach, we have obtained iPS cell lines for several different patients and differentiated
these in vitro into neurons using a protocol that closely mimics human
neuronal development. We are performing immunocytochemistry to determine the
developmental timing of expression of various neuronal markers during differentiation
from both the normal and patient iPS cells and measuring action potentials
and excitatory post-synaptic potentials from these neurons at later times (10 weeks)
of differentiation. Moreover, RNA is being extracted from patient-specific and normal
iPS cells and iPS-derived neurons to produce strand-specific cDNA libraries for
whole transcriptpme (RNA-seq) analysis in order to identify disease-specific gene
networks. Our goal is to model the effects of these specific human gene defects in
vitro in order to study the disease mechanisms and regulation of genomic imprinting
in AS and PWS. The longer term goal of this work is to test small molecules,
environmental modulators, and potential therapies in the human neuronal cell culture
models of AS and PWS.
2455 PATIENT-DERIVED STEM CELLS AS A
TRANSLATIONAL MODEL FOR
NEUROTOXICOLOGICAL RISK.
A. B. Bowman. Neurology, Vanderbilt University, Nashville, TN.
The Bowman lab is developing methods to test the sensitivity of neurons differentiated
from patient-specific induced pluripotent stem cells (iPSCs) to neurotoxicants
associated with neurodegenerative disease. We have generated human iPSCs
from patients having various neurological diseases (e.g. Parkinson’s disease and
Tuberous sclerosis) and controls. These human iPSCs have been differentiated into
neuronal progenitors (evaluated by expression of PAX6 and other neural precursor
markers). These progenitors have subsequently been patterned to specific neuronal
subtypes including dopaminergic (Tryosine Hydroxylase positive) neurons. We
have successfully adapted these neuronal differentiation methods to 96-well plate
format to enable mid to high-throughput gene expression marker analysis by quantitative
RT-PCR as well as cell survival assays for neurotoxicity testing.
Environmentally relevant metal neurotoxicants are being tested for their effect on
these differentiated human neuronal cells, including manganese chloride (50μM to
1000μM) and methyl mercury (100 nM to 10 μM). For example, manganese exposure
(500μM, 20 hours) resulted in complete loss of neuronal processes in
human dopaminergic neurons. Results from these ongoing studies will be presented.
Finally, approaches for efficient neuronal differentiation and toxicological
assessment methodology will be presented – with emphasis on the potential of patient
specific toxicological risk assessment with human iPSCs.
2456 THE USE OF EPIDEMIOLOGICAL DATA AND PBPK
MODELING IN A RISK ASSESSMENT: MANGANESE AS
A CASE STUDY.
H. J. Clewell 1 , M. Andersen 1 , V. Tait 2 , H. Roels 3 and W. Boyes 4 . 1 The Hamner
Institutes for Health Sciences, Research Triangle Park, NC, 2 University of Ottawa,
Ottawa, ON, Canada, 3 Université Catholique de Louvain, Brussels, Belgium and
4 U.S. EPA, Research Triangle Park, NC.
Manganese (Mn) is an essential element that is neurotoxic at high exposures. In recent
years there has been increasing use of PBPK modeling in quantitative risk assessments
to support animal-to-human extrapolation when human risk estimates
are based on animal studies. However, any risk assessment for the neurological effects
of Mn would likely be based on epidemiological data from occupational expo-