Effects of Pesticide Exposure on Stem Cells as a Model for Human Health

The overall goal of the proposed project is to see if adult stem cells will have the capability to differentiate even after short or long periods of exposure to pesticides.There are two objectives:1. The first objective will be to grow/maintain the stem cells, find a suitable dosage or range for pesticide exposure and use various time points for exposure.Stem cells differ from other kinds of cells in the body because they are capable of dividing and renewing themselves for long periods and can give rise to specialized cell types. Stem cells may replicate many times or proliferate therefore producing more stem cells. There are various types of stem cells such as embryonic stem cells (ES), induced pluripotent stem cells (iPSCs) and adult stem cells. Embryonic stem cells (ES) cells are derived from the inner cell mass of the blastocyst, a mainly hollow ball of cells, that forms three to five days after an egg cell is fertilized by a sperm cell in humans. ES cells are pluripotent cells, meaning the cells are able to differentiate into any cell type. These cells are valuable because they provide a renewable resource for studying normal development and disease, and for testing drugs and other therapies. Induced pluripotent stem cells (iPSCs) are pluripotent stem cells that are derived directly from adult tissue. These cells are created from an adult cell such as skin, liver, stomach or other mature cells through the introduction of genes that reprogram the cell and transform it into a cell that has all of the characteristics of an embryonic stem cell. These stem cells are a useful tool for drug development and modeling of diseases. Adult stem cells are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues. These cells can produce different types of cells that maintain the body's tissues and organs as well as the ability to divide and reproduce indefinitely. Since they are able to divide and reproduce indefinitely, their primary role in a living organism is to maintain and repair the tissue in which they are found. Adult stem cells have been found in the brain, bone marrow, blood vessels, skeletal muscle, skin, heart, liver, gut and other organs and tissues. These cells are also referred to as somatic stem cells. Adult stem cells have the ability to differentiate into (1) hematopoietic stem cells which give rise to all the types of blood cells; (2) neural stem cells which are located in the brain and give rise to three major cell types: neurons, astrocytes and oligodendrocytes; (3) mesenchymal stem cells which give rise to a variety of cell types such as bone cells (osteoblasts and osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and stromal cells that support blood formation. Adult stem cells are valuable for transplantation-based therapies. Adult stem cells such as rat fetal neural stem cells will be utilized for this research project as a starting point because of their ability to differentiate into various cell types and their continuous ability to reproduce indefinitely.The first objective is very crucial for the project because specific conditions must be met and maintained at all times in order to expand the cells. One original vial of cells has the potential to make 10 or more vials of cells that can be used for additional experiments. Handling of cells must take place under a hood in a clean, sterile environment. Once the vial of cells is unthawed they are placed in a T-25 flask and are grown and maintained at an appropriate temperature and gas mixture (37 °C, 5% CO2) in a cell incubator. This is essential for the survival of the cells. Depending on the cell type (embryonic, adult, progenitor, induced pluripotent stem cells) the correct cell culture media, like Dulbecco's Modified Eagle's Medium (DMEM) supplemented with animal serum, growth factors, glucose, antibiotics and other nutrients is used to ensure the cells thrive and expand in culture. Some cells require non-standard cell culture reagents, such as conditioned media or feeder cell layers.Adherent and suspension cells are two basic systems for growing cells in culture. Cells that are adherent require an artificial substrate coated with an extracellular matrix component to increase adhesion properties and provide other signals needed for growth and differentiation. Suspension cells are free-floating therefore, the cells don't attach to a surface. If cells are free-floating they are able to grow at a much higher density than adherent cells. The cells must be checked daily for survival/growth and fed every 2-3 days. Once the suspension cells are 80 - 90% confluent in the flask they are harvested and split into 2 flasks. Those 2 flasks then become 4 flasks and so on. At this continuous stage, cells will be harvested and frozen down in liquid nitrogen thus allowing the additional cells to be used for additional experiments. Cells have to continuously be expanded in order to generate more cells, therefore this is why the first objective is so important.2. The second objective is to determine the mortality and ability of stem cells to differentiate after exposure to pesticides important to Kentucky agriculture.Immunocytochemistry (ICC) will be used to determine if cells are capable of differentiation once exposed to pesticides. Immunocytochemistry is a common laboratory technique that is used to assess the presence of a specific protein or antigen in cultured cells or cell suspensions by using a specific antibody, which binds to it, thus allowing visualization and examination under a microscope. The antigen is first bound by a primary antibody which is then amplified by the use of a secondary antibody which binds to the primary antibody. A cytotoxicity assay kit (per established protocols - Promega, Fisher) will be utilized to determine viability of the cells after exposure to pesticides.