Stem Cells - A new chance at life

Cells that have the ability to self-replicate and to give rise to mature cells. The concept of stem cells was originally based on renewing tissues. Many adult tissues, such as the skin, blood, and intestines, consist of mostly mature and short-lived cells that must be continuously replaced. Stem cells were postulated as the source of the self-renewal. In the early 1960s, Canadian scientists Ernest A.

McCulloch and James E. Till provided the first experimental proof of the existence of stem cells in the blood system. They revealed that a type of cell in bone marrow possesses the capacity to replicate itself and to differentiate to various lineages of mature blood cells. Self-renewal, together with the capacity for differentiation, defined the properties of stem cells. This definition is generally used in stem cell biology today.

Stem cells can be found at different stages of fetal development and are present in a wide range of adult tissues. Many of the terms used to distinguish stem cells are based on their origins and the cell types of their progeny. There are three basic types of stem cells. Totipotent stem cells, meaning that their potential is total, have the capacity to give rise to every cell type of the body and to form an entire organism. Pluripotent stem cells, such as embryonic stem cells, are capable of generating virtually all cell types of the body but are unable to form a functioning organism. Multipotent stem cells can give rise only to a limited number of cell types. For example, adult stem cells, also called organ- or tissue-specific stem cells, are multipotent stem cells found in specialized organs and tissues after birth.

Their primary function is to replenish cells lost from normal turnover or disease in the specific organs and tissues in which they are found.

Totipotent and embryonic stem cells.

Totipotent stem cells occur at the earliest stage of embryonic development. The union of sperm and egg creates a single totipotent cell. This cell divides into identical cells in the first hours after fertilization. All these cells have the potential to develop into a fetus when they are placed into the uterus. [To date, no such totipotent stem cell lines (primary cell cultures) have been developed.] The first differentiation of totipotent cells forms a sphere of cells called the blastocyst, which has an outer layer of cells and an inner cell mass. The outer layer of cells will form the placenta and other supporting tissues during fetal development, whereas cells of the inner cell mass go on to form all three primary germ layers: ectoderm, mesoderm, and endoderm. The three germ layers are the embryonic source of all types of cells and tissues of the body.

Embryonic stem cells are de-rivedfrom the inner cell mass of the blastocyst. They retain the capacity to give rise to cells of all three germ layers. However, embryonic stem cells cannot form a complete organism because they are unable to generate the entire spectrum of cells and structures required for fetal development. Thus, embryonic stem cells are pluripotent, not totipotent, stem cells.

Embryonic germ cells.

Embryonic germ cells differ from embryonic stem cells in the tissue sources from which they are derived, but appear to be similar to embryonic stem cells in their pluripotency. Human embryonic germ cell lines are established from the cultures of the primordial germ cells obtained from the gonadal ridge of late-stage embryos, a specific part that normally develops into the testes or the ovaries. Embryonic germ cells in culture, like cultured embryonic stem cells, form embryoid bodies, which are dense, multilayered cell aggregates consisting of partially differentiated cells. The embryoid body-derived cells have high growth potential. The cell lines generated from cultures of the embryoid body cells can give rise to cells of all three embryonic germ layers, indicating that embryonic germ cells may represent another source of pluripotent stem cells.

Growing mouse embryonic stem cells.

Much of the knowledge about embryonic development and stem cells has been accumulated from basic research on mouse embryonic stem cells. The techniques forsep-arating and culturing mouse embryonic stem cells from the inner cell mass of the blastocyst were first developed in the early 1980s. To maintain their growth potential and pluripotency, mouse embryonic stem cells can be grown on a feeder layer, usually consisting of mouse embryonic fibroblast cells. The feeder cells support embryonic stem cells by secreting a cytokine growth factor, the leukemia inhibitory factor, into the growth medium. Alternatively, purified leukemia inhibitory factor can be added to the growth medium without the use of a mouse embryonic feeder layer. (The leukemia inhibitory factor serves as an essential growth factor to maintain embryonic stem cells in culture.) A line of embryonic stem cells can be generated from a single cell under culture conditions that keep embryonic stem cells in a proliferative and undifferentiated state. Embryonic stem cell lines can produce indefinite numbers of identical stem cells. When mouse embryonic stem cells are integrated into an embryo at the blas-tocyst stage, the introduced embryonic stem cells can contribute to cells in all tissues of the resulting mouse. In the absence of feeder cells and the leukemia inhibitory factor in cultures, embryonic stem cells undergo differentiation spontaneously Many studies are focused on directing differentiation of embryonic stem cells in culture. The goal is to generate specific cell types. Formation of cell aggregates with three-dimensional structure during embryonic stem cell differentiation in culture may allow some of the cell-cell interaction to mimic that of in vivo development. The culture conditions can be designed to support and select specific cell types. With these experimental strategies, preliminary success has been achieved to generate some cell types, such as primitive types of vascular structures, blood cells, nerve cells, and pancreatic insulin-producing cells.

Growing human embryonic stem cells.

Since 1998, research teams have succeeded in growing human embryonic stem cells in culture. Human embryonic stem cell lines have been established from the inner cell mass of human blastocysts that were produced through in vitro fertilization procedures. The techniques for growing human embryonic stem cells are similar to those used for growth of mouse embryonic stem cells. However, human embryonic stem cells must be grown on a mouse embryonic fibro-blast feeder layer or in media conditioned by mouse embryonic fibroblasts (see illustration).

There are anumber of human embryonic stem cell lines being generated and maintained in laboratories in the United States and other nations, including Australia, Sweden, India, South Korea, and Israel. The National Institutes of Health has created a Human Embryonic Stem Cell Registry, which lists stem cell lines that have been developed and can be used for research. Human embryonic stem cell lines can be maintained in culture to generate indefinite numbers of identical stem cells for research. As with mouse embryonic stem cells, culture conditions have been designed to direct differentiation into specific cell types (for example, neural and hematopoietic cells).

Adult stem cells.

Adult stem cells, also referred to as somatic stem cells, occur in a wide variety of mature tissues in adults as well as in children. Like all stem cells, adult stem cells can self-replicate. Their ability to self-renew can last throughout the lifetime of individual organisms. Unlike embryonic stem cells, though, it is usually difficult to expand adult stem cells in culture. Adult stem cells reside in specific organs and tissues but account for a very small number of the cells in tissues. They are responsible for maintaining a stable state of the specialized tissues. To replace lost cells, stem cells typically generate intermediate cells called precursor or progenitor cells, which are no longer capable of self-renewal. However, they continue undergoing cell division, coupled with maturation, to yield fully specialized cells. Such stem cells have been identified in many types of adult tissues, including bone mar-row,blood, skin, gastrointestinal tract, dental pulp, retina of the eye, skeletal muscle, liver, pancreas, and brain. Adult stem cells are usually designated according to their source and their potential. Adult stem cells are multipotent because their potential is normally limited to one or more lineages of specialized cells. However, a special multipotent stem cell that can be found in bone marrow, called the mesenchymal stem cell, can produce all cell types of bone, cartilage, fat, blood, and connective tissues.

Blood stem cells.

Blood stem cells, or hematopoietic stem cells, are the most studied type of adult stem cells. The concept of hematopoietic stem cells is not new, as it has been long realized that mature blood cells are constantly lost and destroyed. Billions of new blood cells are produced each day to make up the loss. This process of blood cell generation, called hematopoiesis, occurs largely in the bone marrow. The presence of hematopoietic stem cells in the bone marrow was first demonstrated by E. A. McCulloch and J. E. Till in a mouse model in the early 1960s. The first experimental work on stem cells was an unexpected outcome from their study for measuring the effects of radiation. They found that the blood system of a mouse that has been subjected to heavy radiation can be restored by infusion of bone marrow. The stem cells responsible for reconstituting the blood system generate visible cell colonies on the spleen of the recipient mouse. Each of the spleen colonies consists of one or more types of blood cells, and all the cells in a colony are derived from a single cell. Self-renewal capacity of the colony-forming cells is demonstrated by their ability to form secondary spleen colonies. Such blood stem cells, known as colony forming unit-spleen cells, qualify as pluripotent hematopoi-etic stem cells because they can replicate and give rise to multiple types of mature blood cells. A definitive proof of blood stem cells is their ability to reconstitute the blood system. Bone marrow transplantation demonstrates the restorative powers of blood stem cells in humans.

Isolating blood stem cells.

Like other adult stem cells, blood stem cells are rare and difficult to isolate. Only about1in100,000cellsin the bone marrow is a stem cell. Scientists have used cell-sorting methods to enrich and purify blood stem cells. Stem cells differ from mature cells in their surface markers, which are specific protein molecules on the cell membrane that can be tagged with monoclonal antibodies. By using a set of surface markers, some expressed mainly on stem cells and others on mature blood cells, nearly pure populations of stem cells can be separated from bone marrow. The stem cells purified by this approach can engraft (begin to grow and function) and reconstitute the blood system in the recipient. In animal studies, as few as 30 purified stem cells can rescue a mouse that has been subjected to heavy radiation. Besides the bone marrow, a small number of blood stem cells can be found in circulating blood. In addition, stem cells in the bone marrow can be mobilized into the bloodstream by injecting the donor with certain growth factors or cytokines. This approach can result in a large number of stem cells circulating in peripheral blood, from which they can be collected and used for transplant therapy.
Umbilical cord blood and cord blood banks.

An alternative source of blood stem cells is human umbilical cord blood, a small amount of blood remaining in the placenta and blood vessels of the umbilical cord. It is traditionally treated as a waste material after delivery of the newborn. However, since the recognition of the presence of blood stem cells in umbilical cord blood in the late 1980s, its collection and banking has grown quickly. Similar to bone marrow, umbilical cord blood can be used as a source material of stem cells for transplant therapy. In 1989, the first successful cord blood transplant was reported for treating a 6-year-old boy suffering from Fanconi's anemia (an inherited disease that primarily affects the bone marrow, resulting in decreased production of blood cells) in Paris. Since then, over 6000 cord blood stem cell transplants have been performed worldwide, mainly in patients with blood conditions and in some cancer therapies. However, because of the limited number of stem cells in umbilical cord blood, most ofthe procedures are performed on young children of relatively low body weight. A current focus of study is to promote the growth of umbilical cord blood stem cells in culture in order to generate sufficient numbers of stem cells for adult recipients.

Many blood banks have been established to collect and cryopreserve cord blood cells. Commercial banks offer services of storing cord blood of healthy newborns for potential future use by themselves or their siblings. Although it is considered a biological insurance, the chance ofa child using his or her own cord blood is estimated at 1 per 20,000 collections. Of the estimated 6000 cord blood transplants, only 14 were performed using autologous sources.
Public banks encourage donation of cord blood for unrelated transplants. The Stem Cell Research and Therapeutic Act of 2005 (H.R. 2520) established a national umbilical cord blood program, providing federal funding to collect and store cord blood for blood cell transplants. The program functions to provide a national inventory of 150,000 cord blood units for public use and to establish a registry network integrated with the national marrow donor registry administered by the National Marrow Donor Program (NMDP).

Mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a type of multipotent adult stem cells, and they are defined by the capacity to give rise to a variety of connective tissue lineages, including bone, cartilage, tendon, muscle, and fat cells. Classic studies found a type of cells in bone marrow stroma capa-bleofgenerating fibroblast-like cell colonies. These clonogenic cells were termed colony forming unit-fibroblasts (CFU-F). CFU-F share some characteristics of MSCs. MSCs appear as fibroblast-like spindle-shaped cells. CFU-F assay is still used to evaluate MSCs in cell cultures. MSCs can be distinguished and isolated from other cells based on phenotypic characteristics. Typically, MSCs express specific surface
antigens SH2, SH4, and STRO-1 and lack blood cell markers CD45 and CD34. MSCs can replicate as multipotent cells. The mesenchymal cell lineage potential can be demonstrated in vitro with appropriate culture conditions. Differentiation can be induced to osteocytes by dexamethasone and ascorbate, to chrondrocytes by transforming growth factor-^3, or to adipocytes by dexamethasone and insulin.
MSCs are primarily obtained from bone marrow stromal cells. They are also found in small numbers in umbilical cord blood. In addition, adipose-derived stem cells (ASCs) have been shown to be similar to MSCs. Fat tissue is of mesenchymal origin and contains stromal components. ASCs can be isolated from fat tissue by the method of liposuction. Human ASCs have been shown to exhibit the capacity to give rise to fat, bone, cartilage, muscle, and possibly neurons. Thus, ASCs may provide a potential source of multipotent adult stem cells.
Neural stem cells. Neural stem cells, the multipotent stem cells that generate nerve cells, are a new focus in stem cell research. Active cellular turnover does not occur in the adult nervous system as it does in renewing tissues such as blood or skin. Because of this observation, it had been dogma that the adult brain and spinal cord were unable to regenerate new nerve cells. However, since the early 1990s, neural stem cells have been isolated from the adult brain as well as from fetal brain tissues. Stem cells in the adult brain are found in the areas called the subventricular zone and the ventricle zone. Brain ventricles are small cavities filled with cerebrospinal fluid. Another location of brain stem cells occurs in the hippocampus, a special structure of the cerebral cortex related to memory function. Stem cells isolated from these areas are able to divide and to give rise to nerve cells (neurons) and neuron-supporting cell types in culture.

Plasticity. Stem cell plasticity refers to the phenomenon of adult stem cells from one tissue generating the specialized cells of another tissue. The longstanding concept of adult organ-specific stem cells is that they are restricted to producing the cell types of their specific tissues. However, a series of recent studies have challenged the concept of tissue restriction of adult stem cells. Much of the experimental evidence is derived from transplant studies with blood stem cells. Bone marrow stem cells have been shown to contribute to liver, skeletal muscle, and cardiac cells in human recipients. In mouse models, purified blood stem cells have been demonstrated to generate cells in nonblood tissues, including the liver, gut, and skin. Although the stem cells appear able to cross their tissue-specific boundaries, crossing occurs generally at a low frequency and mostly only under conditions of host organ damage. The finding of stem cell plasticity is unorthodox and unexpected (since adult stem cells are considered to be organ/tissue-specific), but it carries significant implications for potential cell therapy. For example, if differentiation can be redirected, stem cells of abundant source and easy access, such as blood stem cells in bone marrow or umbilical cord blood, could be used to substitute stem cells in tissues that are difficult to isolate, such as heart and nervous system tissue. However, the concept of plasticity has been the subject of controversy.

The observed frequency of lineage conversion is generally low. An alternative explanation to plasticity is the phenomenon of fusion of host and donor cells. Recent findings suggest that blood cells contribute to other tissues by fusing with preexisting cells rather than by converting to other cell lineages.

Potential clinical applications.

Stem cells hold great potential for developing cell therapies to treat a wide range of human diseases. Already in clinical use is blood stem cell transplant therapy, well known as bone marrow transplant therapy for the treatment of patients with certain types of blood diseases and cancers. The discovery of stem cells in various adult tissues, stem cell plasticity, and human embryonic stem cells brings new excitement and opportunities. Stem cells offer the possibility of cell replacement therapy for many human diseases, such as Parkinson's and Alzheimer's diseases, spinal cord injury, diabetes, heart disease, and arthritis, that result from loss or damage of cells in a specialized tissue of the body. Stem cell therapy might revolutionize the treatment and outcome of these diseases. Stem cell science is still in the very early stage. Much more research is required to understand the biological mechanisms that govern cell differentiation and to identify factors that direct cell specialization. Future cell therapy will depend largely on advances in the understanding of stem cell biology and the ability to harness the process of stem cell growth and differentiation. Somatic cell nuclear transfer (SCNT) stem cells.
SCNT involves a micromanipulation procedure in which the nucleus of an egg is removed and replaced by a nucleus taken from somatic cells, typically skin cells. Successful nuclear transfer requires reprogramming of the donor nucleus. The cells so created may divide in cultures to generate embryonic stem cells that can initiate embryogenesis. This is the technique being used in cloning animals, such as the first cloned mammal, Dolly the sheep. However, cloning by nuclear transfer is observed with extremely low efficiency, probably due to faulty and incomplete reprogramming of the donor nucleus. The mechanisms governing the transition from a differentiated genome to a totipotent state remain largely unknown.
A major interest in SCNT is the prospect of creating patient-specific embryonic stem cells. These cells would be genetically identical to the nuclear donor except for maternal mitochondrial deoxyribonucleic acid (mtDNA) of the oocyte. Therefore, the problem of graft rejection would be avoided if the cells could be used for transplant therapy for the donor patients. The concept of using SCNT to generate customized stem cells for cell therapy is also referred to as therapeutic cloning. However, there are hurdles and limitations to using embryonic stem cells in clinical applications. A major challenge is to achieve the directed differentiation and controlled growth before stem cells can be used for transplant therapy. Another issue on SCNT in human stem cells is the sourcing of human eggs. The procedure requires a large number of eggs from women, and poses an ethical and technical challenge.

The success in producing embryonic stem cell lines by the SCNT technique has been demonstrated in mice. In an article published in Science in 2005, a team led by Hwang Woo Suk of South Korea claimed the establishment of patient-specific stem cell lines by using the SCNT technique. However, the paper was later retracted as the results were fabricated and the claim a fraud. The field is still left uncertain if somatic nuclear replacement is feasible in humans.
Ethical and regulatory issues. The use of human embryonic stem cells raises ethical, social, and legal issues. The major concern centers on the source of stem cells. Human embryonic stem cell lines are made from the inner cell mass of a blastocyst stage embryo. Most embryos used to produce stem cells are left over from in vitro fertilization (TVF) treatment. The embryos are destroyed by the procedure of extracting stem cells. The early embryo has the biological potential to develop into a person. However, society has not reached consensus on when human life begins. The attention on stem cell research and cloning calls for regulation and legislation from governments. In the United States, current policy allows federal funds to be used for research only on existing human embryonic stem lines. The human embryonic stem cell lines that meet the eligibility criteria are listed in the Human Embryonic Stem Cell Registry by the National Institutes of Health (NIH).

One concern about SCNT is that it may lead to the reproductive cloning of humans. In theory, the embryo created via SCNT could be used to clone a human if it were implanted into a womans uterus. In the United States, the legislators in the House of Representatives and the Senate have introduced bills proposing a ban of all forms of cloning, including research cloning, or inhibiting reproductive cloning while preserving therapeutic cloning research. However, these bills have not been passed, and no federal law has been established on human embryonic stem cell research. The Canadian Parliament has passed Bill C-6 that prohibits creation of a human clone, sale of sperm or ova, and commercial surrogacy. The bill permits the use of stem cells obtained from discarded products of in vitro fertilization, that is, excess and unused embryos. In the United Kingdom, a law permits the use of embryos in research and therapeutic cloning research but bans reproductive cloning, and implanting a cloned embryo in a human uterus is liable to criminal prosecution. Cloning research must be licensed from the Human Fertilization and Embryology Authority that governs embryonic and stem cell research in the United Kingdom.
Reference : McGraw - Hill Encyclopedia of Science and Technology


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