STEM CELL TECHNOLOGY

Stem cells are defined as totipotent progenitor cells capable of self renewal and multilineage differentiation. Stem cells survive well and show stable division in culture, making them ideal targets for in vitro manipulation. Stem cell technology is a rapidly developing field that combines the efforts of cell biologists, geneticists, and clinicians and offers hope of effective treatment for a variety of malignant and non-malignant diseases. Stem cells are distinguished from other cell types by their capacity of renewing themselves through cell division, sometimes after long period of inactivity and under certain physiologic or experimental conditions, stem cells can be induced to become tissue- or organ-specific cells with special functions. The role of stem cells is particularly evident in the case of several types of differentiated cells, including blood cells, epithelial cells of the skin, and the epithelial cells lining the digestive tract, that have short life spans and must be replaced by continual cell proliferation in adult animals. The production of blood cells provides a good example of the role of stem cells in maintaining differentiated cell populations. All of these blood cells have limited life spans and are continually produced by the division of a pluripotent stem cell in the bone marrow (Figure-1). Descendants of the pluripotent stem cell then continue to proliferate and undergo several rounds of divisions as they differentiate.

Figure 1: Development of different types of blood cells from a pluripotent stem cell in the bone marrow

Embryonic stem cells

Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of the blastocyst, an earlystage embryo. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ

layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. Sexual reproduction begins when a male's sperm fertilizes a female's ovum (egg) to form a single cell called a zygote. The single zygote cell then begins a series of divisions, forming 2, 4, 8, 16 cells, etc. After four to six days, before implantation in the uterus, this mass of cells is called a blastocyst. The blastocyst consists of an inner cell mass (embryoblast) and an outer cell mass (trophoblast). The outer cell mass becomes part of the placenta, and the inner cell mass is the group of cells that will differentiate to become all the structures of an adult organism. This latter mass is the source of embryonic stem cells - totipotent cells (cells with total potential to develop into any cell in the body). Nearly all research to date has made use of mouse embryonic stem cells (mES) (Figure-2) or human embryonic stem cells (hES). Both have the essential stem cell characteristics, yet they require very different environments in order to maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatin and require the presence of leukemia inhibitory factor (LIF). Human ES cells are grown on a feeder layer of mouse embryonic fibroblasts (MEFs) and require the presence of basic fibroblast growth factor (bFGF or FGF-2). ES cells, being pluripotent cells, require specific signals for correct differentiation; if injected directly into another body, ES cells will differentiate into many different types of cells, causing a teratoma.

Figure 2: Mouse embryonic stem cells with fluorescent marker

Adult stem cells

Adult or somatic stem cells exist throughout the body after embryonic development and are found inside of different types of tissue. These stem cells have been found in tissues such as the brain, bone marrow, blood, blood vessels, skeletal muscles, skin, and the liver. They remain in a quiescent or non-dividing state for years until activated by disease or tissue injury. Adult stem cells can divide or self-renew indefinitely, enabling them to generate a range of cell types from the originating organ or even regenerate the entire original organ. It is generally thought that adult stem cells are limited in their ability to differentiate based on their tissue of origin, but there is some evidence to suggest that they can differentiate to become other cell types. Adult stem cells, and tissues derived from them, are currently believed less likely to initiate rejection after transplantation. This is because a patient's own cells could be expanded in culture, coaxed into assuming a specific cell type (differentiation), and then reintroduced into the patient. Adult stem cell treatments have been successfully used for many years to treat leukemia and related bone/blood cancers through bone marrow transplants. Adult stem cells are also used in veterinary medicine to treat tendon and ligament injuries in horses.

Amniotic stem cells

Amniotic stem cells found in amniotic fluid are very active, expand extensively without feeders and are not tumorigenic. Amniotic stem cells are multipotent and can differentiate in cells of adipogenic, osteogenic, myogenic, endothelial, hepatic and also neuronal lines. Use of stem cells from amniotic fluid overcomes the ethical objections to using human embryos as a source of cells.

Induced pluripotent stem cells (iPSCs)

Induced pluripotent stem cells (iPSCs): Induced pluripotent stem cells are adult cells that have been genetically reprogrammed to an embryonic stem celllike state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Mouse iPSCs were first reported in 2006, and human iPSCs were first reported in late 2007. Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including expressing stem cell markers, forming tumors containing cells from all three germ layers, and being able to contribute to many different tissues when injected into mouse embryos at a very early stage in development. Human iPSCs also express stem cell markers and are capable of generating cells characteristic of all three germ layers. Induced pulripotent stem cells are useful tools for drug development and modelling of diseases, and scientists hope to use them in transplantation medicine. Viruses are currently used to introduce the reprogramming factors into adult cells. Researchers are currently investigating non-viral delivery strategies in animal studies, as virus used to introduce the stem cell factors sometimes causes cancers. In any case, this breakthrough discovery has created a powerful new way to "de-differentiate" cells whose developmental fates had been previously assumed to be determined. In addition, tissues derived from iPSCs will be a nearly identical match to the cell donor and thus probably avoid rejection by the immune system.

Stem cell cultures

Stem cells are either extracted from adult tissue or from a dividing zygote in a culture dish. Once extracted, the cells are placed in a controlled culture that prohibits them from further specializing or differentiating but usually allows them to divide and replicate. The process of growing large numbers of embryonic stem cells has been easier than growing large numbers of adult stem cells. Embryonic stem cells of mice are pluripotent cultured cells derived from early preimplantation embryos. These cells can be maintained and multiplied in vitro long enough to permit the various manipulations for gene transfer. The cultured pluripotent embryonic stem cells are transfected with the appropriate transgene construct by a suitable transfection technique. Transfected embryonic stem cells are identified and selected, by employing a selectable marker gene, and are cloned.

Stem cell lines

After the division and propagation of stem cells in a controlled culture, the collection of healthy, dividing, and undifferentiated cells is called a stem cell line. Embryonic stem cells are able to differentiate into more cell types than adult stem cells. To ensure self-renewal, stem cells undergo two types of cell division, symmetric division gives rise to two identical daughter cells both endowed with stem cell properties. Asymmetric division, on the other hand, produces only one stem cell and a progenitor cell with limited self-renewal potential. Progenitors can go through several rounds of cell division before terminally differentiating into a mature cell. It is possible that the molecular distinction between symmetric and asymmetric divisions lies in differential segregation of cell membrane proteins (such as receptors) between the daughter cells.

Stem cell therapy

Medical researchers believe that stem cell therapy has the potential to dramatically change the treatment of human disease. Because of the replication and differentiation ability of stem cells, they are of considerable interest with respect to potential medical applications. The stem cells with the broadest differentiative capacity are the embryonic stem cells that are present in early embryos and can give rise to all of the differentiated cell types of adult organisms. The isolation of human embyonic stem cells followed the first demonstration, in 1997; that the nucleus of an adult cell could give rise to a viable cloned animal following transplantation into an oocyte. Transfer of nuclei from adult somatic cells into oocytes has subsequently been used to create cloned offspring of a variety of species including sheep,

mice, pigs, cattle, goats, rabbits and cats. In therapeutic cloning, a nucleus from an adult human cell would be transferred to an oocyte, which would be used to produce an early embryo in culture. Embryonic stem cells cultured from such a cloned embryo could then, in principle, be used to generate appropriate types of differentiated cells for transplantation therapy. The possibility of such transplantation therapy offers hope for the treatment of a variety of devasting disorders, including Parkinson's disease, Alzheimer's disease, diabetes and spinal cord injuries. A number of adult stem cell therapies already exist, particularly bone marrow transplants that are used to treat leukemia.Substantial improvements are meeded in the methods used to generate embryos by nuclear transfer and it will be necessary to develop procedures that reliably differentiate embyonic stem cells into the appropriate cell types prior to transplantation. The development of therapeutic cloning also raises ethical concerns, not only with respect to possibility of cloning human beings, but also with respect to the destruction of embryos that serve as the source of embryonic stem cells. While many challenges remain, continuing research on stem cells holds great promise of opening new approaches to the treatment of a broad array of human diseases.