Nagina Gilani 10-arid-1768 Ph.D Zoology. Introduction of stem cells Characteristics Formation and differentiation Types Stem cell culture Applications.

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Presentation transcript:

Nagina Gilani 10-arid-1768 Ph.D Zoology

Introduction of stem cells Characteristics Formation and differentiation Types Stem cell culture Applications of stem cells in toxicological assays

A cell that has the ability to continuously divide and differentiate (develop) into various other kind(s) of cells/tissues

‘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

Formation and 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.

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

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.

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

 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

Cyto-toxicity assays developmental toxicity assays cell functional assays

Cytotoxicity is the quality of being toxic to cells Cytotoxicity results Cells necrosis (Cell lysis) Activates cell apoptosis

Effectors Macrophages and Granulocytes Natural Killer (NK) cells Cytotoxic T lymphocytes (CTL) Target cells Virally infected cells Cells infected with intracellular bacteria

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

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

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

Effectors Radiation Infections Maternal metabolic imbalances Drugs and chemicals Target cells Brain, eyes, heart, skeletal muscles etc.

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)

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.

Function of mature cells differentiated from stem cells (ES cells or iPS cells ) Inhibitory Stimulatory Metabolic and electrophysiological functions of somatic cells are measured

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

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

Some terms related to electrophysiology are, Electrocardiography (heart) Electroencephalography (brain) Electromyography (muscles) Electro-oculography (eyes) Electro-retinography- (retina) Audiology (auditory system)

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

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

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

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

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.

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.

Hideki Mori and Masayuki Hara Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka , Japan Received 17 August 2012; accepted 20 May 2013 Available online 1 July 2013