CULTURED STEM CELLS AS TOOLS FOR

TOXICOLOGICAL ASSAYS

Nagina Gilani10-arid-1768Ph.D Zoology

Introduction of stem cells

Characteristics

Formation and differentiation

Types

Stem cell culture

Applications of stem cells in toxicological assays

Contents

A cell that has the ability to continuously divide and

differentiate (develop) into various other kind(s) of

cells/tissues

STEM CELL

‘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

Formation and differentiation

Stem Cell Differentiation

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

Stem cells culture

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

Mouse fibroblasts

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

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

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…

Cyto-toxicity assays

developmental toxicity assays

cell functional assays

Stem cell-based toxicological assays

Stem cells in toxicological assays

Cytotoxicity is the quality of being toxic to cells

Cytotoxicity results

Cells necrosis (Cell lysis)

Activates cell apoptosis

Cyto-toxicity(Cell killing or lysis)

Effectors

Macrophages and Granulocytes

Natural Killer (NK) cells

Cytotoxic T lymphocytes (CTL)

Target cells

Virally infected cells

Cells infected with intracellular bacteria

Cyto-toxicity

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

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…

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

Effectors

Radiation

Infections

Maternal metabolic imbalances

Drugs and chemicals

Target cells

Brain, eyes, heart, skeletal muscles etc.

Developmental toxicity

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

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…

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

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

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…

Some terms related to electrophysiology are,

Electrocardiography (heart)

Electroencephalography (brain)

Electromyography (muscles)

Electro-oculography (eyes)

Electro-retinography- (retina)

Audiology (auditory system)

Continued…

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

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

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…

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

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…

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

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

THANKS