主題一: Stem Cell的定義

摘錄自: Stem Cell Information (The official National Institutes of Health resource for stem cell research)

I. What are stem cells and why are they important?

Stem cells have two important characteristics that distinguish them from other types of cells. First, they are unspecialized cells that renew themselves for long periods through cell division. The second is that under certain physiologic or experimental conditions, they can be induced to become cells with special functions such as the beating cells of the heart muscle or the insulinproducing cells of the pancreas.
Scientists primarily work with two kinds of stem cells from animals and humans: embryonic stem cells and adult stem cells, which have different functions and characteristics that will be explained in this document. Scientists discovered ways to obtain or derive stem cells from early mouse embryos more than 20 years ago. Many years of detailed study of the biology of mouse stem cells led to the discovery, in 1998, of how to isolate stem cells from human embryos and grow the cells in the laboratory. These are called human embryonic stem cells. The embryos used in these studies were created for infertility purposes through in vitro fertilization procedures and when they were no longer needed for that purpose, they were donated for research with the informed consent of the donor.
Stem cells are important for living organisms for many reasons. In the 3- to 5-day-old embryo, called a blastocyst, stem cells in developing tissues give rise to the multiple specialized cell types that make up the heart, lung, skin, and other tissues. In some adult tissues, such as bone marrow, muscle, and brain, discrete populations of adult stem cells generate replacements for cells that are lost through normal wear and tear, injury, or disease.
It has been hypothesized by scientists that stem cells may, at some point in the future, become the basis for treating diseases such as Parkinson's disease, diabetes, and heart disease.
Scientists want to study stem cells in the laboratory so they can learn about their essential properties and what makes them different from specialized cell types. As scientists learn more about stem cells, it may become possible to use the cells not just in cell-based therapies, but also for screening new drugs and toxins and understanding birth defects. However, as mentioned above, human embryonic stem cells have only been studied since 1998. Therefore, in order to develop such treatments scientists are intensively studying the fundamental properties of stem cells, which include: 1. determining precisely how stem cells remain unspecialized and self renewing for many years; and 2. identifying the signals that cause stem cells to become specialized cells.

II. What are the unique properties of all stem cells?

Stem cells differ from other kinds of cells in the body. All stem cells—regardless of their source—have three general properties: they are capable of dividing and renewing themselves for long periods; they are unspecialized; and they can give rise to specialized cell types.
Scientists are trying to understand two fundamental properties of stem cells that relate to their longterm self-renewal: 1. why can embryonic stem cells proliferate for a year or more in the laboratory without differentiating, but most adult stem cells cannot; and 2. what are the factors in living organisms that normally regulate stem cell proliferation and self-renewal?
Discovering the answers to these questions may make it possible to understand how cell proliferation is regulated during normal embryonic development or during the abnormal cell division that leads to cancer. Importantly, such information would enable scientists to grow embryonic and adult stem cells more efficiently in the laboratory.
Stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. A stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell); it cannot carry molecules of oxygen through the bloodstream (like a red blood cell); and it cannot fire electrochemical signals to other cells that allow the body to move or speak (like a nerve cell). However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.
Stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cells—which do not normally replicate themselves—stem cells may replicate many times. When cells replicate themselves many times over it is called proliferation. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be unspecialized, like the parent stem cells, the cells are said to be capable of long-term self-renewal.
The specific factors and conditions that allow stem cells to remain unspecialized are of great interest to scientists. It has taken scientists many years of trial and error to learn to grow stem cells in the laboratory without them spontaneously differentiating into specific cell types. For example, it took 20 years to learn how to grow human embryonic stem cells in the laboratory following the development of conditions for growing mouse stem cells. Therefore, an important area of research is understanding the signals in a mature organism that cause a stem cell population to proliferate and remain unspecialized until the cells are needed for repair of a specific tissue. Such information is critical for scientists to be able to grow large numbers of unspecialized stem cells in the laboratory for further experimentation.
Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation. Scientists are just beginning to understand the signals inside and outside cells that trigger stem cell differentiation. The internal signals are controlled by a cell's genes, which are interspersed across long strands of DNA, and carry coded instructions for all the structures and functions of a cell. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment.
Therefore, many questions about stem cell differentiation remain. For example, are the internal and external signals for cell differentiation similar for all kinds of stem cells? Can specific sets of signals be identified that promote differentiation into specific cell types? Addressing these questions is critical because the answers may lead scientists to find new ways of controlling stem cell differentiation in the laboratory, thereby growing cells or tissues that can be used for specific purposes including cell-based therapies.
Adult stem cells typically generate the cell types of the tissue in which they reside. A blood-forming adult stem cell in the bone marrow, for example, normally gives rise to the many types of blood cells such as red blood cells, white blood cells and platelets. Until recently, it had been thought that a blood-forming cell in the bone marrow—which is called a hematopoietic stem cell—could not give rise to the cells of a very different tissue, such as nerve cells in the brain. However, a number of experiments over the last several years have raised the possibility that stem cells from one tissue may be able to give rise to cell types of a completely different tissue, a phenomenon known as plasticity. Examples of such plasticity include blood cells becoming neurons, liver cells that can be made to produce insulin, and hematopoietic stem cells that can develop into heart muscle. Therefore, exploring the possibility of using adult stem cells for cell-based therapies has become a very active area of investigation by researchers.

III. What are embryonic stem cells?

A. What stages of early embryonic development are important for generating embryonic stem cells?
Embryonic stem cells, as their name suggests, are derived from embryos. Specifically, embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitro—in an in vitro 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. The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst. The blastocyst includes three structures: the trophoblast, which is the layer of cells that surrounds the blastocyst; the blastocoel, which is the hollow cavity inside the blastocyst; and the inner cell mass, which is a group of approximately 30 cells at one end of the blastocoel.
B. How are embryonic stem cells grown in the laboratory?
Growing cells in the laboratory is known as cell culture. Human embryonic stem cells are isolated by transferring the inner cell mass 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. The inner surface of the culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide. This coating layer of cells is called a feeder layer. The reason for having the mouse cells in the bottom of the culture dish is to give the inner cell mass cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium. Recently, scientists have begun to devise ways of growing embryonic stem cells without the mouse feeder cells. This is a significant scientific advancement because of the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells.
Over the course of several days, the cells of the inner cell mass proliferate and begin to crowd the culture dish. When this occurs, they are removed gently and plated into several fresh culture dishes. The process of replating the cells is repeated many times and for many months, and is called subculturing. Each cycle of subculturing the cells is referred to as a passage. After six months or more, the original 30 cells of the inner cell mass yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for six or more months without differentiating, are pluripotent, and appear genetically normal are referred to as an embryonic stem cell line. Once cell lines are established, or even before that stage, batches of them can be frozen and shipped to other laboratories for further culture and experimentation.
C. What laboratory tests are used to identify embryonic stem cells?
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.
As yet, scientists who study human embryonic stem cells have not agreed on a standard battery of tests that measure the cells' fundamental properties. Also, scientists acknowledge that many of the tests they do use may not be good indicators of the cells' most important biological properties and functions. Nevertheless, laboratories that grow human embryonic stem cell lines use several kinds of tests. These tests include:
Growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term 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 surface markers that are found only on undifferentiated cells. Another important test is for the presence of a protein called Oct-4, which undifferentiated cells typically make. Oct-4 is a transcription factor, meaning that it helps turn genes on and off at the right time, which is an important part of the processes of cell differentiation and embryonic development.
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 subcultured after freezing, thawing, and replating.
Testing whether the human embryonic stem cells are pluripotent by 1) allowing the cells to differentiate spontaneously in cell culture; 2) manipulating the cells so they will differentiate to form specific cell types; or 3) injecting the cells into an immunosuppressed mouse to test for the formation of a benign tumor called a teratoma. 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.
D. How are embryonic stem cells stimulated to differentiate?
As long as the embryonic stem cells in culture are grown under certain 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. Through years of experimentation scientists have established some basic protocols or "recipes" for the directed differentiation of embryonic stem cells into some specific cell types.
If scientists can reliably direct the differentiation of embryonic stem cells into specific cell types, they may be able to use the resulting, differentiated cells to treat certain diseases at some point in the future. Diseases that might be treated by transplanting cells generated from human embryonic stem cells include Parkinson's disease, diabetes, traumatic spinal cord injury, Purkinje cell degeneration, Duchenne's muscular dystrophy, heart disease, and vision and hearing loss.
Stem Cell Basics

IV. What are adult stem cells?

An adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ, can renew itself, and can differentiate to yield 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. Some scientists now use the term somatic stem cell instead of adult stem cell.
Unlike embryonic stem cells, which are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is unknown.
Research on adult stem cells has recently generated a great deal of excitement. Scientists have found adult stem cells in many more tissues than they once thought possible. This finding has led scientists to ask whether adult stem cells could be used for transplants. In fact, adult blood forming stem cells from bone marrow have been used in transplants for 30 years. Certain kinds of adult stem cells seem to have the ability to differentiate into a number of different cell types, given the right conditions. If this differentiation of adult stem cells can be controlled in the laboratory, these cells may become the basis of therapies for many serious common diseases.
The history of research on adult stem cells began about 40 years ago. In the 1960s, 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 cells, was discovered a few years later. Stromal cells are a mixed cell population that generates bone, cartilage, fat, and fibrous connective tissue.
Also in the 1960s, scientists who were studying rats discovered two regions of the brain that contained dividing cells, which become nerve cells. Despite these reports, most scientists believed that new nerve cells could not be generated in the adult brain. 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—astrocytes and oligodendrocytes, which are non-neuronal cells, and neurons, or nerve cells.
A. Where are adult stem cells found and what do they normally do?
Adult stem cells have been identified in many organs and tissues. One important point to understand about adult stem cells is that there are a very small number of stem cells in each tissue. Stem cells are thought to reside in a specific area of each tissue where they may remain quiescent (non-dividing) for many years until they are activated by disease or tissue injury. The adult tissues reported to contain stem cells include brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin and liver.
Scientists in many laboratories are trying to find ways to grow adult stem cells in cell culture and manipulate them to generate specific cell types so they can be used to treat injury or disease. Some examples of potential treatments include replacing the dopamine-producing cells in the brains of Parkinson's patients, developing insulin-producing cells for type I diabetes and repairing damaged heart muscle following a heart attack with cardiac muscle cells.
B. What tests are used for identifying adult stem cells?
Scientists do not agree on the criteria that should be used to identify and test adult stem cells.
However, they often use one or more of the following three methods: (1) labeling the cells in a living tissue with molecular markers and then determining the specialized cell types they generate; (2) removing the cells from a living animal, labeling them in cell culture, and transplanting them back into another animal to determine whether the cells repopulate their tissue of origin; and (3) isolating the cells, growing them in cell culture, and manipulating them, often by adding growth factors or introducing new genes, to determine what differentiated cells types they can become.
Also, a single adult stem cell should be able to generate a line of genetically identical cells—known as a clone—which then gives rise to all the appropriate differentiated cell types of the tissue. a clone—which then gives rise to all the appropriate differentiated cell types of the tissue. Scientists tend to show either that a stem cell can give rise to a clone of cells in cell culture, or that a purified population of candidate stem cells can repopulate the tissue after transplant into an animal. Recently, by infecting adult stem cells with a virus that gives a unique identifier to each individual cell, scientists have been able to demonstrate that individual adult stem cell clones have the ability to repopulate injured tissues in a living animal.
C. What is known about adult stem cell differentiation?
As indicated above, scientists have reported that adult stem cells occur in many tissues and that they enter normal differentiation pathways to form the specialized cell types of the tissue in which they reside. Adult stem cells may also exhibit the ability to form specialized cell types of other tissues, which is known as transdifferentiation or plasticity.
Normal differentiation pathways of adult stem cells. In a living animal, adult stem cells can divide for a long period and can give rise to mature cell types that have characteristic shapes and specialized structures and functions of a particular tissue. The following are examples of differentiation pathways of adult stem cells.
Hematopoietic stem cells give rise to all the types of blood cells: red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and platelets.
Bone marrow stromal cells (mesenchymal stem cells) give rise to a variety of cell types: bone cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and other kinds of connective tissue cells such as those in tendons.
Neural stem cells in the brain give rise to its three major cell types: nerve cells (neurons) and two categories of non-neuronal cells—astrocytes and oligodendrocytes.
Epithelial stem cells in the lining of the digestive tract occur in deep crypts and give rise to several cell types: absorptive cells, goblet cells, Paneth cells, and enteroendocrine cells.
Skin stem cells occur in the basal layer of the epidermis and at the base of hair follicles. The epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer. The follicular stem cells can give rise to both the hair follicle and to the epidermis.
Adult stem cell plasticity and transdifferentiation. A number of experiments have suggested that certain adult stem cell types are pluripotent. This ability to differentiate into multiple cell types is called plasticity stem cell types are pluripotent. This ability to differentiate into multiple cell types is called plasticity or transdifferentiation. The following list offers examples of adult stem cell plasticity that have been reported during the past few years.
Hematopoietic stem cells may differentiate into: three major types of brain cells (neurons, oligodendrocytes, and astrocytes); skeletal muscle cells; cardiac muscle cells; and liver cells.
Bone marrow stromal cells may differentiate into: cardiac muscle cells and skeletal muscle cells.
Brain stem cells may differentiate into: blood cells and skeletal muscle cells.
Current research is aimed at determining the mechanisms that underlie adult stem cell plasticity. If such mechanisms can be identified and controlled, existing stem cells from a healthy tissue might be induced to repopulate and repair a diseased tissue.
D. What are the key questions about adult stem cells?
Many important questions about adult stem cells remain to be answered. They include:
How many kinds of adult stem cells exist, and in which tissues do they exist?
What are the sources of adult stem cells in the body? Are they "leftover" embryonic stem cells, or do they arise in some other way? Why do they remain in an undifferentiated state when all the cells around them have differentiated?
Do adult stem cells normally exhibit plasticity, or do they only transdifferentiate when scientists manipulate them experimentally? What are the signals that regulate the proliferation and differentiation of stem cells that demonstrate plasticity?
Is it possible to manipulate adult stem cells to enhance their proliferation so that sufficient tissue for transplants can be produced?
Does a single type of stem cell exist—possibly in the bone marrow or circulating in the blood—that can generate the cells of any organ or tissue?
What are the factors that stimulate stem cells to relocate to sites of injury or damage?

V. What are the similarities and differences between embryonic and adult stem cells?
Human embryonic and adult stem cells each have advantages and disadvantages regarding potential use for cell-based regenerative therapies. Of course, adult and embryonic stem cells differ in the number and type of differentiated cells types they can become. Embryonic stem cells can become all cell types of the body because they are pluripotent. Adult stem cells are generally limited to differentiating into different cell types of their tissue of origin. However, some evidence suggests that adult stem cell plasticity may exist, increasing the number of cell types a given adult stem cell can become.
Large numbers of embryonic stem cells can be relatively easily grown in culture, while adult stem cells are rare in mature tissues and methods for expanding their numbers in cell culture have not yet been worked out. This is an important distinction, as large numbers of cells are needed for stem cell replacement therapies.
A potential advantage of using stem cells from an adult is that the patient's own cells could be expanded in culture and then reintroduced into the patient. The use of the patient's own adult stem cells would mean that the cells would not be rejected by the immune system. This represents a significant advantage as immune rejection is a difficult problem that can only be circumvented with immunosuppressive drugs.
Embryonic stem cells from a donor introduced into a patient could cause transplant rejection. However, whether the recipient would reject donor embryonic stem cells has not been determined in human experiments.



主題二: Stem Cell Markers

摘錄自: USA | (800) 343-7475 2008 R&D Systems


STEM CELL MARKERS
* Embryonic Stem Cell Markers
* Hematopoietic Stem Cell Markers
* Mesenchymal/Stromal Stem Cell Markers
* Neural Stem Cell Markers
   
While stem cells are best defined functionally, a number of molecular markers have been used to characterize various stem cell populations.

Although functions have yet to be ascertained for many of these early markers, their unique expression pattern and timing provide a useful tool for scientists to initially identify as well as isolate stem cells. This mini-review summarizes evidence regarding the roles of specific markers in defining embryonic, hematopoietic, mesenchymal/stromal, and neural stem cell populations. For most of the molecules discussed, studies performed both in vitro and in vivo support their significant role in characterizing stem cells. Until more is known about the novel marker-negative stem cell population, however, uncertainty still exists regarding the benefits of using these markers alone or in various combinations when identifying and isolating cells for stem cell research.

Embryonic Stem Cell Markers
Oct-4: Oct-4 (also termed Oct-3 or Oct3/4), one of the POU transcription factors, was originally identified as a DNA-binding protein that activates gene transcription via a cis-element containing octamer motif. It is expressed in totipotent embryonic stem and germ cells. A critical level of Oct-4 expression is required to sustain stem cell self-renewal and pluripotency. Differentiation of embryonic stem (ES) cells results in down- regulation of Oct-4, an event essential for a proper and divergent developmental program. Oct-4 is not only a master regulator of pluripotency that controls lineage commitment, but is also the first and most recognized marker used for the identification of totipotent ES cells.
SSEAs (Stage Specific Embryonic Antigens): SSEAs were originally identified by three monoclonal antibodies (Abs) recognizing defined carbohydrate epitopes associated with lacto- and globo-series glycolipids, SSEA-1, -3 and - 4. SSEA-1 is expressed on the surface of preimplantation-stage murine embryos (i.e. at the eight cell stage) and has been found on the surface of teratocarcinoma stem cells, but not on their differentiated derivatives. The oviduct epithelium, endometrium and epididymis, as well as some areas of the brain and kidney tubules in adult mice have also been shown to be reactive with SSEA-1 Abs. SSEA-3 and -4 are synthesized during oogenesis and are present in the membranes of oocytes, zygotes and early cleavage-stage embryos. Biological roles of these carbohydrate-associated molecules have been suggested in controlling cell surface interactions during development. Undifferentiated primate ES cells, human EC and ES cells express SSEA-3 and SSEA-4, but not SSEA-1. Undifferentiated mouse ES cells express SSEA-1, but not SSEA-3 or SSEA-4.

Hematopoietic Stem Cell Markers
CD34: The cell surface sialomucin CD34 has been a focus of interest ever since it was found expressed on a small fraction of human bone marrow cells. The CD34+-enriched cell population from marrow or mobilized peripheral blood appears responsible for most of the hematopoietic activity. CD34 has therefore been considered to be the most critical marker for hematopoietic stem cells (HSCs). CD34 expression on primitive cells is down-regulated as they differentiate into mature cells. It is also found on clonogenic progenitors, however, and some lineage-committed cells. Although its precise function is still unknown, the pattern of expression of CD34 suggests that it plays a significant role in early hematopoiesis. The theory of CD34 being the most primitive HSC marker, however, has recently been challenged. Osawa et al. first demonstrated that murine HSCs could be CD34 negative. In addition, a low level of engraftment and hematopoietic capacity has been demonstrated in human CD34- cells. Transplantation studies also showed repopulating activity in a CD34- cell population in fetal sheep. Additionally, studies have shown that both murine and human CD34+ cells may be derived from CD34- cells. Collectively, these reports suggest the possibility that HSCs may be CD34+ or CD34- and that selection of cells expressing CD34 might result in exclusion of more primitive stem cells. Nevertheless, almost all clinical and experimental protocols including ex vivo culture, gene therapy, and HSC transplantation are currently designed for cell populations enriched for CD34+ cells.
CD133: CD133, a 120 kDa, glycosylated protein containing five transmembrane domains, was identified initially by the AC133 monoclonal Ab, which recognizes a CD34+ subset of human HSCs. A CD133 isoform, AC133-2, has been recently cloned and identified as the original surface antigen recognized by the AC133 Ab. CD133 may provide an alternative to CD34 for HSC selection and ex vivo expansion. A CD133+ enriched subset can be expanded in a similar manner as a CD34+ enriched subset, retaining its multilineage capacity. Recent studies have offered evidence that CD133 expression is not limited to primitive blood cells, but defines unique cell populations in non-hematopoietic tissues as well. CD133+ progenitor cells from peripheral blood can be induced to differentiate into endothelial cells in vitro. In addition, human neural stem cells can be directly isolated by using an anti-CD133 Ab.
ABCG2: ABCG2 (ATP-binding cassette superfamily G member 2) is a determinant of the Hoechst-negative phenotype of side population (SP) cells and found in a wide variety of stem cells, including HSC. ABCG2 is a member of the family of ABC transporters and was first identified in a breast cancer cell line. It belongs to the half-transporter group and is unique as it is localized to the plasma membrane. The expression of ABCG2 appears greatest on CD34- cells and is down-regulated with the acquisition of CD34 on the cell surface. Down-regulation in ABCG2 expression is also observed in various committed hematopoietic progenitors. ABCG2 may therefore serve as a more promising marker than CD34 for primitive HSC isolation and characterization. The expression pattern of ABCG2, however, is not limited to HSC. ABCG2 expression exclusively characterizes the Hoechst SP phenotype in cells from diverse sources, including monkey bone marrow, mouse skeletal muscle and ES cells. The potential plasticity of SP cells has been demonstrated by studies showing that cardiomyocytes and muscle can be regenerated from transplanted bone marrow-derived SP cells. Exclusive expression of ABCG2 on SP cells suggests that ABCG2 may be a potential marker for positive selection of pluripotent stem cells from various adult sources. ABCG2 has been implicated in playing a functional role in developmental stem cell biology.
Sca-1: Sca-1 (stem cell antigen 1, Ly-6A/E), an 18 kDa phosphatidylinositol-anchored protein, is a member of the Ly-6 antigen family. Sca-1 is the most recognized HSC marker in mice with both Ly-6 haplotypes as it is expressed on multipotent HSCs. An anti-Sca-1 Ab is frequently used in combination with negative selection for expression of a number of cell surface markers characteristic of differentiated cells of hematolymphoid lineages (Lin-) to identify and isolate murine HSCs. Sca-1+ HSCs can be found in the adult bone marrow, fetal liver and mobilized peripheral blood and spleen within the adult animal. Sca-1 has also been discovered in several non-hematopoietic tissues, however, and can be used to enrich progenitor cell populations other than HSCs. Sca-1 may be involved in regulating both B and T cell activation.

Mesenchymal/Stromal Stem Cell Markers
STRO-1: The murine IgM monoclonal Ab STRO-1, produced from an immunization with a population of human CD34+ bone marrow cells, can identify a cell surface antigen expressed by stromal elements in human bone marrow. From bone marrow cells, the frequency of fibroblast colony-forming cells (CFU-F) is enriched approximately 100-fold in the STRO-1+/Glycophorin A- population than in the STRO-1+/Glycophorin A+ population. A STRO-1+ enriched subset of marrow cells is capable of differentiating into multiple mesenchymal lineages including hematopoiesis-supportive stromal cells with a vascular smooth muscle-like phenotype, adipocytes, osteoblasts and chondrocytes. STRO-1 is a valuable Ab for the identification, isolation and functional characterization of human bone marrow stromal cell precursors, which are quite distinct from those of primitive HSCs.

Neural Stem Cell Markers
Nestin: Nestin is a class VI intermediate filament protein. Although it is expressed predominantly in stem cells of the central nervous system (CNS), its expression is absent from nearly all mature CNS cells. Nestin has been the most extensively used marker to identify CNS stem cells within various areas of the developing nervous system and in cultured cells in vitro. The role of nestin in CNS stem cell biology, however, remains undefined. Although nestin does not form intermediate filaments by itself in vitro it does co-assemble with vimentin or alpha-internexin to form and heterodimer, coiled-coil complexes that may then form intermediate filaments. Its transient expression has been suggested to be a major step in the neural differentiation pathway. Nestin expression has also been discovered in non-neural stem cell populations, such as pancreatic islet progenitors as well as hematopoietic progenitors.
PSA-NCAM (Polysialic acid-neural cell adhesion molecule): The regulated expression of neural cell adhesion molecule (NCAM) isoforms in the brain is critical for many neural developmental processes. The embryonic form of NCAM, PSA-NCAM, is highly polysialylated and is mainly expressed in the developing nervous system. PSA-NCAM may be related to synaptic rearrangement and plasticity. In the adult, PSA-NCAM expression is restricted to regions that retain plasticity. A neuronal-restricted precursor identified by its high expression of PSA-NCAM can undergo self-renewal and differentiate into multiple neuronal phenotypes. PSA-NCAM+ neonatal brain precursors are restricted to a glial fate and thyroid hormone can modulate them into an oligodendrocyte fate. Polysialic acid modification significantly decreases NCAM adhesiveness and therefore, it was originally suggested PSA-NCAM works as a purely anti-adhesive factor that modulates cell-cell interactions in promoting brain plasticity. Increasing evidence indicates that PSA-NCAM may interact with secreted signaling molecules to perform an instructive role in development.
p75 Neurotrophin R (NTR): p75 NTR, also named low affinity nerve growth factor (NGF) receptor, is a type I transmembrane protein that belongs to the tumor necrosis factor receptor superfamily. It binds to NGF, BDNF, NT-3 and NT-4 equally (with low affinity). p75NTR, when activated in the presence of Trk, enhances responses to neurotrophin. TrkC receptors working together with p75 NTR have been suggested to serve critical functions during the development of the nervous system. Neural crest stem cells (NCSCs) have been isolated based on their surface expression of p75NTR. Freshly isolated p75NTR+ NCSCs from peripheral nerve tissues can self-renew and generate neurons and glia both in vitro and in vivo. In addition, neuroepithelial-derived p75NTR+ cells are also able to differentiate into neurons, smooth muscle and Schwann cells in culture. Recently, p75 NTR has been used as a marker to identify mesenchymal precursors as well as hepatic stellate cells.

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