i. Embryonic Stem cells:
Embryonic stem cells are derived from embryos. Most embryonic stem cells are derived from embryos that develop from eggs that have been fertilized invitro in an invitro fertilization clinic and then donated for research purposes with informed consent of the donors. They are not derived from eggs fertilized in a woman's body.
Growing cells in the laboratory is known as cell culture. Human embryonic stem cells are generated by transferring cells from a pre implantation stage embryo into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The cells divide and spread over the surface of the dish. In the original protocol, the inner surface of the culture dish was coated with mouse embryonic skin cells specially treated so they will not divide. This coating layer of cells is called a feeder layer. The mouse cells in the bottom of the culture dish provide the cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium. Researchers have now devised ways to grow embryonic stem cells without mouse feeder cells. This is a significant scientific advance because of the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells.
The process of generating an embryonic stem cell line is somewhat inefficient, so lines are not produced each time cells from the pre implantation stage embryo are placed into a culture dish. However, if the plated cells survive, divide and multiply enough to crowd the dish, they are removed gently and plated into several fresh culture dishes. The process of re-plating or subculturing the cells is repeated many times and for many months. Each cycle of sub culturing the cells is referred to as a passage. Once the cell line is established, the original cells yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for a prolonged period of time without differentiating, and are pluripotent are referred to as an embryonic stem cell line. At any stage in the process, batches of cells can be frozen and shipped to other laboratories for further culture and experimentation.
Characterization of embryonic cell:
At various points during the process of generating embryonic stem cell lines, scientists test the cells to see whether they exhibit the fundamental properties that make them embryonic stem cells. This process is called characterization.
Scientists who study human embryonic stem cells have not yet agreed on a standard battery of tests that measure the cells' fundamental properties.
However, laboratories that grow human embryonic stem cell lines use several kinds of tests, including:
* Growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term growth and self-renewal. Scientists inspect the cultures through a microscope to see that the cells look healthy and remain undifferentiated.
* Using specific techniques to determine the presence of transcription factors that are typically produced by undifferentiated cells. Two of the most important transcription factors are Nanog and Oct 4. Transcription factors help turn genes on and off at the right time, which is an important part of the processes of cell differentiation and embryonic development. In this case, both Oct 4 and Nanog are associated with maintaining the stem cells in an undifferentiated state, capable of self-renewal.
* Using specific techniques to determine the presence of particular cell surface markers that are typically produced by undifferentiated cells.
* Examining the chromosomes under a microscope. This is a method to assess whether the chromosomes are damaged or if the number of chromosomes has changed. It does not detect genetic mutations in the cells.
* Determining whether the cells can be re-grown, or subcultured, after freezing, thawing, and re-plating.
* Testing whether the human embryonic stem cells are pluripotent by
a) Allowing the cells to differentiate spontaneously in cell culture.
b) Manipulating the cells so they will differentiate to form cells characteristic of the three germ layers.
c) Injecting the cells into a mouse with a suppressed immune system to test for the formation of a benign tumor called a teratoma. Since the mouse’s immune system is suppressed, the injected human stem cells are not rejected by the mouse immune system and scientists can observe growth and differentiation of the human stem cells. Teratomas typically contain a mixture of many differentiated or partly differentiated cell types an indication that the embryonic stem cells are capable of differentiating into multiple cell types.
Embryonic Stem cells differentiate to other cells:
As long as the embryonic stem cells in culture are grown under appropriate conditions, they can remain undifferentiated (unspecialized). But if cells are allowed to clump together to form embryoid bodies, they begin to differentiate spontaneously.
They can form muscle cells, nerve cells, and many other cell types. Although spontaneous differentiation is a good indication that a culture of embryonic stem cells is healthy, it is not an efficient way to produce cultures of specific cell types.
So, to generate cultures of specific types of differentiated cells heart muscle cells, blood cells, or nerve cells, for example scientists try to control the differentiation of embryonic stem cells. They change the chemical composition of the culture medium, alter the surface of the culture dish, or modify the cells by inserting specific genes.
ii. Adult stem cells:
An adult stem cell is thought to be an undifferentiated cell, found among differentiated cells in a tissue or organ. The adult stem cell can renew itself and can differentiate to yield some or all of the major specialized cell types of the tissue or organ. The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found. Scientists also use the term somatic stem cell instead of adult stem cell, where somatic refers to cells of the body. Unlike embryonic stem cells, which are defined by their origin (cells from the pre implantation-stage embryo), the origin of adult stem cells in some mature tissues is still under investigation.
The history of research on adult stem cells began more than 60 years ago. In the 1950s, researchers discovered that the bone marrow contains at least two kinds of stem cells. One population, called hematopoietic stem cells, forms all the types of blood cells in the body. A second population called bone marrow stromal stem cells, were discovered a few years later. These non-hematopoietic stem cells make up a small proportion of the stromal cell population in the bone marrow and can generate bone, cartilage, and fat cells that support the formation of blood and fibrous connective tissue.
In the 1960s, scientists who were studying rats discovered two regions of the brain that contained dividing cells that ultimately become nerve cells. Despite these reports, most scientists believed that the adult brain could not generate new nerve cells. It was not until the 1990s that scientists agreed that the adult brain does contain stem cells that are able to generate the brain's three major cell types are astrocytes and oligodendrocytes, which are non neuronal cells, and neurons, or nerve cells.
Principle of Adult Stem cells:
Adult stem cells have been identified in many organs and tissues, including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, and testis.
They are thought to reside in a specific area of each tissue. In many tissues, current evidence suggests that some types of stem cells are pericytes, cells that compose the outermost layer of small blood vessels. Stem cells may remain quiescent for long periods of time until they are activated by a normal need for more cells to maintain tissues, or by disease or tissue injury.
Typically, there is a very small number of stem cells in each tissue and, once removed from the body, their capacity to divide is limited, making generation of large quantities of stem cells difficult. Scientists in many laboratories are trying to find better ways to grow large quantities of adult stem cells in cell culture and to manipulate them to generate specific cell types so they can be used to treat injury or disease. Some examples of potential treatments include regenerating bone using cells derived from bone marrow stroma, developing insulin-producing cells for type 1 diabetes, and repairing damaged heart muscle following a heart attack with cardiac muscle cells.
Tests used to identify adult stem cells:
Scientists often use one or more of the following methods to identify adult stem cells:
a) Label the cells in a living tissue with molecular markers and then determine the specialized cell types they generate
b) Remove the cells from a living animal, label them in cell culture, and transplant them back into another animal to determine whether the cells replace their tissue of origin.
iii. Difference between embryonic and adult stem cells:
* Embryonic stem cells can become all cell types of the body because they are pluripotent.
* Adult stem cells are thought to be limited to differentiating into different cell types of their tissue of origin.
* Embryonic stem cells can be grown relatively easily in culture.
* Adult stem cells are rare in mature tissues, so isolating these cells from an adult tissue is challenging, and methods to expand their numbers in cell culture have not yet been worked out.
Scientists believe that tissues derived from embryonic and adult stem cells may differ in the likelihood of being rejected after transplantation. We don't yet know for certain whether tissues derived from embryonic stem cells would cause transplant rejection, since relatively few clinical trials have tested the safety of transplanted cells derived from human embroyonic stem cells.
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. The use of adult stem cells and tissues derived from the patient's own adult stem cells would mean that the cells are less likely to be rejected by the immune system. This represents a significant advantage, as immune rejection can be circumvented only by continuous administration of immunosuppressive drugs, and the drugs themselves may cause deleterious side effects.
iv. Induced pluripotent stem cells:
Induced pluripotent stem cells are adult cells that have been genetically reprogrammed to an embryonic stem cell like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells. Although these cells meet the defining criteria for pluripotent stem cells, it is not known if induced Pluripotent Stem Cells (iPSCs) and embryonic stem cells differ in clinically significant ways. 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.
Although additional research is needed, iPSCs are already useful tools for drug development and modeling of diseases, and scientists hope to use them in transplantation medicine. Viruses are currently used to introduce the reprogramming factors into adult cells, and this process must be carefully controlled and tested before the technique can lead to useful treatment for humans. In animal studies, the virus used to introduce the stem cell factors sometimes causes cancers. Researchers are currently investigating non-viral delivery strategies. 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. The iPSC strategy creates pluripotent stem cells that, together with studies of other types of pluripotent stem cells, will help researchers learn how to reprogram cells to repair damaged tissues in the human body.
v. Limbal Stem cell:
Limbal stem cells are stem cells located in the basal epithelial layer of the corneal limbus.
Characteristics of limbal stem:
* Slow turnover rate
* High proliferative potential
* Expression of stem cell markers
* The ability to regenerate the entire corneal epithelium.
* Proliferation of limbal stem cells maintains the cornea; for example, replacing cells that are lost via tears. Limbal stem cells also prevent the conjunctival epithelial cells from migrating onto the surface of the cornea.
Damage to the limbus can lead to limbal stem cell deficiency (LSCD), which can be caused by burns, radiation, genetic disorders, surgeries, infection, use of contact lenses, or drug use. Signs and symptoms include conjunctivalisation, corneal vascularisation, edema, ocular discomfort or pain, poor vision, and blindness, which are likely associated with failure in the process of regenerating the corneal epithelium.
Most treatments for LSCD are palliative, but severe LSCD due to burns can be treated by stem cell-based regenerative medicine. In 2015, the European Commission approved a stem cell therapy for people with severe LSCD due to burns, which was the first time that a stem cell therapy other than the use of umbilical cord stem cells was allowed to be sold by any regulatory agency in the world.