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CULTURED STEM CELLS AS TOOLS FOR
TOXICOLOGICAL ASSAYS
Nagina Gilani10-arid-1768Ph.D Zoology
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Introduction of stem cells
Characteristics
Formation and differentiation
Types
Stem cell culture
Applications of stem cells in toxicological assays
Contents
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A cell that has the ability to continuously divide and
differentiate (develop) into various other kind(s) of
cells/tissues
STEM CELL
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‘Blank cells’ (unspecialized)
Have the potential to give rise to specialized types of
cell (differentiation)
Capable of dividing and renewing themselves for
long periods of time (proliferation and renewal)
Ability to repair in the living organism
CHARACTERISTICS
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Formation and differentiation
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Stem Cell Differentiation
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Embryonic stem cells (from a 5-6 day-old embryo):
They have the ability to form virtually any type of cell
found in the human body.
Embryonic germ cells (from late embryo or fetus) will
ultimately produce eggs or sperm (gametes).
Adult stem cells (undifferentiated cells found among
specialized or differentiated cells) in a tissue or organ
after birth.
Types of Stem Cells
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Stem cells culture
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Conditioning means culturing of stem cells (ES and
iPS) under ideal conditions
Mouse fibroblasts are used as feeder layers in many
studies but it may causes problems
Feeder free culture systems using extracellular
matrices (ECMs) and other effective compounds
have been developed
Importance of conditioning for stem cell culture
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Mouse fibroblasts
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A toxin is a poisonous substance produced within
living cells or organisms e.g small
molecules, peptides.
Toxicity is the degree to which a substance can
damage an organism.
Toxicology concerned with the study of the adverse
effects of chemicals on living organisms.
Toxins, toxicity and toxicology
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A toxicity assay is carried out by using terminally
differentiated cells but is difficult for live animals
because of the varying absorption and distribution
parameters in live animals
Whole embryo culture and harvesting of human adult
tissue is not possible with human embryos, for ethical
reasons
Importance of the stem cell assay system in
toxicology
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Gap between the cell-based toxicological assays and
assays using experimental animals, is narrowed by
tissue engineering technologies by producing artificial
3D tissues for toxicological assay using cells derived
from pluripotent stem cells
The effect of the analyte substance on cell
differentiation and tissue plasticity is determined
Continued…
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Cyto-toxicity assays
developmental toxicity assays
cell functional assays
Stem cell-based toxicological assays
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Stem cells in toxicological assays
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Cytotoxicity is the quality of being toxic to cells
Cytotoxicity results
Cells necrosis (Cell lysis)
Activates cell apoptosis
Cyto-toxicity(Cell killing or lysis)
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Effectors
Macrophages and Granulocytes
Natural Killer (NK) cells
Cytotoxic T lymphocytes (CTL)
Target cells
Virally infected cells
Cells infected with intracellular bacteria
Cyto-toxicity
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Embryonic stem cell (ESC) and Somatic stem cells
(SSCs) are used
Metal ions or xenobiotics in human body or central
nervous system (CNS) evaluated by EST
Metals used as biomaterials in medical devices are
evaluated by mesenchymal and neural stem cells
Cyto-toxicity assays
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The European Center for the Validation of Alternative
Methods (ECVAM), describes the embryonic stem
cell test (EST) to investigate toxicity
Half survival concentration (IC50) value of a
substance is determined by viable cell count and cell
survival rate
Continued…
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The adverse effects on a developing organism as a
result of exposure to a chemical before conception, in
utero, after birth, or during sexual maturation
Results
Abortions
Premature births
Abnormal development of organs
Developmental toxicity
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Effectors
Radiation
Infections
Maternal metabolic imbalances
Drugs and chemicals
Target cells
Brain, eyes, heart, skeletal muscles etc.
Developmental toxicity
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Abnormal differentiation of stem cells into other cell
types, developmental abnormalities and
Carcinogenesis due to abnormal development of stem
cells is evaluated
Pluripotent and somatic stem cells (neural and
mesenchymal) are used (cyto-toxicity assay and test
for differentiation potential are combined)
Developmental toxicity assays
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The IC50 (50% survival concentration) value
measured in fibroblasts and ES cells
The ID50 value (50% inhibition of ES cell
differentiation) is measured
Neuro-developmental toxicity assays, environmental
toxicity testing, and screening of new drugs for CNS
diseases are still under development.
Continued…
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Function of mature cells differentiated from stem
cells (ES cells or iPS cells )
Inhibitory
Stimulatory
Metabolic and electrophysiological functions of
somatic cells are measured
Cell functional assays
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Hepatocytes are having drug metabolizing
capacities
They are used in drug screening protocols
Metabolically functioning hepatocytes generated
for albumin secretion and glycogen storage in
liver
Cell functional assays
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Neurons and myocardial cells are used for
electrophysiological measurements
Myocardial cells are generated and cultured on
surface of a multi-electrode measurement system to
monitor the action potential generation
Continued…
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Some terms related to electrophysiology are,
Electrocardiography (heart)
Electroencephalography (brain)
Electromyography (muscles)
Electro-oculography (eyes)
Electro-retinography- (retina)
Audiology (auditory system)
Continued…
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Heavy metal ions (lead and mercury) are produced by
pollution cause inhibition of trace elements into cells
Zinc (Zn) toxicity (an essential micronutrient that can
affect somatic stem cells)
Somatic and neural stem cells are used
Assessment of heavy metal ions
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Micro-electrical/mechanical systems (MEMS), tip
technologies, and bio-sensing are used for
manipulating and evaluating cells properties and
functions in culture
Cell morphology imaging, cyto-toxicity assays,
fluorescent microscopy are used to determine
physiological state of cells
Combined use of toxicological assay with cell tips and micro reactors
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These technologies helps in generation of 3D tissue-
like structures to prolong the viability of artificial
tissues in culture (vascular tissue)
It is helpful to evaluate the risk of toxic substances to
living organisms
Continued…
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Valid and reliable toxicological assay system using
stem cells can be used in the risk assessment of
pollutants for public safety
An assay system for evaluating toxicity should be
simple, easy to use for use as a standard method
Conclusion
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The protocol for testing of embryonic toxicity using
ES cells is published by ECVAM to be widely used
as a standard method for evaluating developmental
toxicity with modifications depending on the tissues
being tested.
Continued…
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It will be possible to evaluate the specific potential
risk of a toxic substance before body is exposed to
substance.
Prevention of toxicity is based on knowledge of
molecular mechanisms of toxin action
More collaborative studies are needed to develop
standardized methodologies for use in toxicology
assessment systems.
Future perspectives
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Hideki Mori and Masayuki Hara Department of
Biological Science, Graduate School of Science,
Osaka Prefecture University, 1-2 Gakuen-cho, Naka-
ku, Sakai, Osaka 599 8570, Japan Received 17 August
2012; accepted 20 May 2013 Available online 1 July
2013
Reference
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THANKS