Stem Cell Basics

1. Introduction: What are stem cells, and why are they important? 2. What are the unique properties of all stem cells? 3. What are embryonic stem cells? 4. What are adult stem cells? 5. What are the similarities and differences between embryonic and adult stem cells? 6. What are induced pluripotent stem cells? 7. What are the potential uses of human stem cells and the obstacles that must be overcome before these potential uses will be

realized? 8. Where can I get more information?

I. Introduction: What are stem cells, and why are they important?

Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell.

Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.

Until recently, scientists primarily worked with two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic "somatic" or "adult" stem cells. The functions and characteristics of these cells will be explained in this document. Scientists discovered ways to derive embryonic stem cells from early mouse embryos nearly 30 years ago, in 1981. The detailed study of the biology of mouse stem cells led to the discovery, in 1998, of a method to derive stem cells from human embryos and grow the cells in the laboratory. These cells are called human embryonic stem cells. The embryos used in these studies were created for reproductive purposes through in vitro fertilization procedures. When they were no longer needed for that purpose, they were donated for research with the informed consent of the donor. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be "reprogrammed" genetically to assume a stem cell-like state. This new type of stem cell, called induced pluripotent stem cells (iPSCs), will be discussed in a later section of this document.

Stem cells are important for living organisms for many reasons. In the 3- to 5-day-old embryo, called a blastocyst, the inner cells give rise to the entire body of the organism, including all of the many specialized cell types and organs such as the heart, lung, skin, sperm, eggs 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.

Given their unique regenerative abilities, stem cells offer new potentials for treating diseases such as diabetes, and heart disease. However, much work remains to be done in the laboratory and the clinic to understand how to use these cells for cell-based therapies to treat disease, which is also referred to as regenerative or reparative medicine.

Laboratory studies of stem cells enable scientists to learn about the cells’ essential properties and what makes them different from specialized cell types. Scientists are already using stem cells in the laboratory to screen new drugs and to develop model systems to study normal growth and identify the causes of birth defects.

Research on stem cells continues to advance knowledge about how an organism develops from a single cell and how healthy cells replace damaged cells in adult organisms. Stem cell research is one of the most fascinating areas of contemporary biology, but, as with many expanding fields of scientific inquiry, research on stem cells raises scientific questions as rapidly as it generates new discoveries.

I. Introduction 

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.

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, or proliferate. 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.

Scientists are trying to understand two fundamental properties of stem cells that relate to their long-term self-renewal:

1. why can embryonic stem cells proliferate for a year or more in the laboratory without differentiating, but most non-embryonic 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. Such information would also enable scientists to grow embryonic and non-embryonic stem cells more efficiently in the laboratory.

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 derive and maintain stem cells in the laboratory without them spontaneously differentiating into specific cell types. For example, it took two decades to learn how to grow human embryonic stem cells in the laboratory following the development of conditions for growing mouse stem cells. Therefore, understanding the signals in a mature organism that cause a stem cell population to proliferate and remain unspecialized until the cells are needed. 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 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. For example, a stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell), and it cannot carry oxygen molecules through the bloodstream (like a red blood cell). However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.

Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation. While differentiating, the cell usually goes through several stages, becoming more specialized at each step. Scientists are just beginning to understand the signals inside and outside cells that trigger each stem of the differentiation process. The internal signals are controlled by a cell's genes, which are interspersed across long strands of DNA, and carry coded instructions for all cellular structures and functions. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment. The interaction of signals during differentiation causes the cell's DNA to acquire epigenetic marks that restrict DNA expression in the cell and can be passed on through cell division.

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 may lead scientists to find new ways to control stem cell differentiation in the laboratory, thereby growing cells or tissues that can be used for specific purposes such as cell-based therapies or drug screening.

Adult stem cells typically generate the cell types of the tissue in which they reside. For example, a blood-forming adult stem cell in the bone marrow normally gives rise to the many types of blood cells. It is generally accepted that a blood-forming cell in the bone marrow—which is called a hematopoietic stem cell—cannot give rise to the cells of a very different tissue, such as nerve cells in the brain. Experiments over the last several years have purported to show that stem cells from one tissue may give rise to cell types of a completely different tissue. This remains an area of great debate within the research community. This controversy demonstrates the challenges of studying adult stem cells and suggests that additional research using adult stem cells is necessary to understand their full potential as future therapies.

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. Most 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.

B. How are embryonic stem cells grown in the laboratory?

Growing cells in the laboratory is known as cell culture. Human embryonic stem cells (hESCs) are generated by transferring cells from a preimplantation-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. 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 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 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 preimplantation-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 subculturing 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 for a prolonged period of time without differentiating, are pluripotent, and have not developed genetic abnormalities 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.

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.

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 Oct4. 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 paricular 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 1) allowing the cells to differentiate spontaneously in cell

culture; 2) manipulating the cells so they will differentiate to form cells characteristic of the three germ layers; or 3) 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.

D. How are embryonic stem cells stimulated to differentiate?

Figure 1. Directed differentiation of mouse embryonic stem cells. Click here for larger image. (© 2001 Terese Winslow)

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. 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 (Figure 1). (For additional examples of directed differentiation of embryonic stem cells, refer to the NIH stem cell reports available at /info/2006report/ and /info/2001report/2001report.htm.)

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 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, Duchenne's muscular dystrophy, heart disease, and vision and hearing loss.

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IV. What are adult stem cells?

An adult stem cell is thought to be an undifferentiated cell, found among differentiated cells in a tissue or organ that 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 (not the germ cells, sperm or eggs). Unlike embryonic stem cells, which are defined by their origin (cells from the preimplantation-stage embryo), the origin of adult stem cells in some mature tissues is still under investigation.

Research on adult stem cells has 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 researchers and clinicians to ask whether adult stem cells could be used for transplants. In fact, adult hematopoietic, or blood-forming, stem cells from bone marrow have been used in transplants for 40 years. Scientists now have evidence that stem cells exist in the brain and the heart. If the differentiation of adult stem cells can be controlled in the laboratory, these cells may become the basis of transplantation-based therapies.

The history of research on adult stem cells began about 50 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 (also called mesenchymal stem cells, or skeletal stem cells by some), 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, 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—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, 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 (called a "stem cell niche"). 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 (non-dividing) 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.

B. What tests are used for identifying adult stem cells?

Scientists often use one or more of the following methods to identify adult stem cells: (1) label the cells in a living tissue with molecular markers and then determine the specialized cell types they generate; (2) 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 (or "repopulate") their tissue of origin.

Importantly, it must be demonstrated that a single adult stem cell can generate a line of genetically identical cells that then gives rise to all the appropriate differentiated cell types of the tissue. To confirm experimentally that a putative adult stem cell is indeed a stem cell,

scientists tend to show either that the cell can give rise to these genetically identical cells in culture, and/or that a purified population of these candidate stem cells can repopulate or reform the tissue after transplant into an animal.

C. What is known about adult stem cell differentiation?

Figure 2. Hematopoietic and stromal stem cell differentiation. Click here for larger image. (© 2001 Terese Winslow)

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.

Normal differentiation pathways of adult stem cells. In a living animal, adult stem cells are available to divide, when needed, 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 (Figure 2) that have been demonstrated in vitro or in vivo.

← 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, and macrophages.

← 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.

Transdifferentiation. A number of experiments have reported that certain adult stem cell types can differentiate into cell types seen in organs or tissues other than those expected from the cells' predicted lineage (i.e., brain stem cells that differentiate into blood cells or blood-forming cells that differentiate into cardiac muscle cells, and so forth). This reported phenomenon is called transdifferentiation.

Although isolated instances of transdifferentiation have been observed in some vertebrate species, whether this phenomenon actually occurs in humans is under debate by the scientific community. Instead of transdifferentiation, the observed instances may involve fusion of a donor cell with a recipient cell. Another possibility is that transplanted stem cells are secreting factors that encourage the recipient's own

stem cells to begin the repair process. Even when transdifferentiation has been detected, only a very small percentage of cells undergo the process.

In a variation of transdifferentiation experiments, scientists have recently demonstrated that certain adult cell types can be "reprogrammed" into other cell types in vivo using a well-controlled process of genetic modification (see Section VI for a discussion of the principles of reprogramming). This strategy may offer a way to reprogram available cells into other cell types that have been lost or damaged due to disease. For example, one recent experiment shows how pancreatic beta cells, the insulin-producing cells that are lost or damaged in diabetes, could possibly be created by reprogramming other pancreatic cells. By "re-starting" expression of three critical beta-cell genes in differentiated adult pancreatic exocrine cells, researchers were able to create beta cell-like cells that can secrete insulin. The reprogrammed cells were similar to beta cells in appearance, size, and shape; expressed genes characteristic of beta cells; and were able to partially restore blood sugar regulation in mice whose own beta cells had been chemically destroyed. While not transdifferentiation by definition, this method for reprogramming adult cells may be used as a model for directly reprogramming other adult cell types.

In addition to reprogramming cells to become a specific cell type, it is now possible to reprogram adult somatic cells to become like embryonic stem cells (induced pluripotent stem cells, iPSCs) through the introduction of embryonic genes. Thus, a source of cells can be generated that are specific to the donor, thereby avoiding issues of histocompatibility, if such cells were to be used for tissue regeneration. However, like embryonic stem cells, determination of the methods by which iPSCs can be completely and reproducibly committed to appropriate cell lineages is still under investigation.

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?

← How do adult stem cells evolve during development and how are they maintained in the adult? Are they "leftover" embryonic stem cells, or do they arise in some other way?

← Why do stem cells remain in an undifferentiated state when all the cells around them have differentiated? What are the characteristics of their “niche” that controls their behavior?

← Do adult stem cells have the capacity to transdifferentiate, and is it possible to control this process to improve its reliability and efficiency?

← If the beneficial effect of adult stem cell transplantation is a trophic effect, what are the mechanisms? Is donor cell-recipient cell contact required, secretion of factors by the donor cell, or both?

← What are the factors that control adult stem cell proliferation and differentiation? ← What are the factors that stimulate stem cells to relocate to sites of injury or damage, and how can this process be enhanced for

better healing?

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. One major difference between adult and embryonic stem cells is their different abilities in the number and type of differentiated cell types they can become. 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. This is an important distinction, as large numbers of cells are needed for stem cell replacement therapies.

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 whether tissues derived from embryonic stem cells would cause transplant rejection, since the first phase 1 clinical trials testing the safety of cells derived from hESCS have only recently been approved by the United States Food and Drug Administration (FDA).

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

VI. What are induced pluripotent stem cells?

Induced pluripotent stem cells (iPSCs) 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 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 treatments 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.

VII. What are the potential uses of human stem cells and the obstacles that must be overcome before these potential uses will be realized?

There are many ways in which human stem cells can be used in research and the clinic. Studies of human embryonic stem cells will yield information about the complex events that occur during human development. A primary goal of this work is to identify how undifferentiated stem cells become the differentiated cells that form the tissues and organs. Scientists know that turning genes on and off is central to this process. Some of the most serious medical conditions, such as cancer and birth defects, are due to abnormal cell division and differentiation. A more complete understanding of the genetic and molecular controls of these processes may yield information about how such diseases arise and suggest new strategies for therapy. Predictably controlling cell proliferation and differentiation requires additional basic research on the molecular and genetic signals that regulate cell division and specialization. While recent developments with iPS cells suggest some of the specific factors that may be involved, techniques must be devised to introduce these factors safely into the cells and control the processes that are induced by these factors.

Human stem cells could also be used to test new drugs. For example, new medications could be tested for safety on differentiated cells generated from human pluripotent cell lines. Other kinds of cell lines are already used in this way. Cancer cell lines, for example, are used to screen potential anti-tumor drugs. The availability of pluripotent stem cells would allow drug testing in a wider range of cell types. However, to screen drugs effectively, the conditions must be identical when comparing different drugs. Therefore, scientists will have to be able to precisely control the differentiation of stem cells into the specific cell type on which drugs will be tested. Current knowledge of the signals controlling differentiation falls short of being able to mimic these conditions precisely to generate pure populations of differentiated cells for each drug being tested.

Perhaps the most important potential application of human stem cells is the generation of cells and tissues that could be used for cell-based therapies. Today, donated organs and tissues are often used to replace ailing or destroyed tissue, but the need for transplantable tissues and organs far outweighs the available supply. Stem cells, directed to differentiate into specific cell types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases including Alzheimer's diseases, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis.

Figure 3. Strategies to repair heart muscle with adult stem cells. Click here for larger image.

© 2001 Terese Winslow

For example, it may become possible to generate healthy heart muscle cells in the laboratory and then transplant those cells into patients with chronic heart disease. Preliminary research in mice and other animals indicates that bone marrow stromal cells, transplanted into a damaged heart, can have beneficial effects. Whether these cells can generate heart muscle cells or stimulate the growth of new blood vessels that repopulate the heart tissue, or help via some other mechanism is actively under investigation. For example, injected cells may accomplish repair by secreting growth factors, rather than actually incorporating into the heart. Promising results from animal studies have served as the basis for a small number of exploratory studies in humans (for discussion, see call-out box, "Can Stem Cells Mend a Broken Heart?"). Other recent studies in cell culture systems indicate that it may be possible to direct the differentiation of embryonic stem cells or adult bone marrow cells into heart muscle cells (Figure 3).

Can Stem Cells Mend a Broken Heart?: Stem Cells for the Future Treatment of Heart Disease

Cardiovascular disease (CVD), which includes hypertension, coronary heart disease, stroke, and congestive heart failure, has ranked as the number one cause of death in the United States every year since 1900 except 1918, when the nation struggled with an influenza epidemic. Nearly 2600 Americans die of CVD each day, roughly one person every 34 seconds. Given the aging of the population and the relatively dramatic recent increases in the prevalence of cardiovascular risk factors such as obesity and type 2 diabetes, CVD will be a significant health concern well into the 21st century.

Cardiovascular disease can deprive heart tissue of oxygen, thereby killing cardiac muscle cells (cardiomyocytes). This loss triggers a cascade of detrimental events, including formation of scar tissue, an overload of blood flow and pressure capacity, the overstretching of viable cardiac cells attempting to sustain cardiac output, leading to heart failure, and eventual death. Restoring damaged heart muscle tissue, through repair or regeneration, is therefore a potentially new strategy to treat heart failure.

The use of embryonic and adult-derived stem cells for cardiac repair is an active area of research. A number of stem cell types, including embryonic stem (ES) cells, cardiac stem cells that naturally reside within the heart, myoblasts (muscle stem cells), adult bone marrow-derived cells including mesenchymal cells (bone marrow-derived cells that give rise to tissues such as muscle, bone, tendons, ligaments, and adipose tissue), endothelial progenitor cells (cells that give rise to the endothelium, the interior lining of blood vessels), and umbilical cord blood cells, have been investigated as possible sources for regenerating damaged heart tissue. All have been explored in mouse or rat models, and some have been tested in larger animal models, such as pigs.

A few small studies have also been carried out in humans, usually in patients who are undergoing open-heart surgery. Several of these have demonstrated that stem cells that are injected into the circulation or directly into the injured heart tissue appear to improve cardiac function and/or induce the formation of new capillaries. The mechanism for this repair remains controversial, and the stem cells likely regenerate

heart tissue through several pathways. However, the stem cell populations that have been tested in these experiments vary widely, as do the conditions of their purification and application. Although much more research is needed to assess the safety and improve the efficacy of this approach, these preliminary clinical experiments show how stem cells may one day be used to repair damaged heart tissue, thereby reducing the burden of cardiovascular disease.

In people who suffer from type 1 diabetes, the cells of the pancreas that normally produce insulin are destroyed by the patient's own immune system. New studies indicate that it may be possible to direct the differentiation of human embryonic stem cells in cell culture to form insulin-producing cells that eventually could be used in transplantation therapy for persons with diabetes.

To realize the promise of novel cell-based therapies for such pervasive and debilitating diseases, scientists must be able to manipulate stem cells so that they possess the necessary characteristics for successful differentiation, transplantation, and engraftment. The following is a list of steps in successful cell-based treatments that scientists will have to learn to control to bring such treatments to the clinic. To be useful for transplant purposes, stem cells must be reproducibly made to:

← Proliferate extensively and generate sufficient quantities of tissue.

← Differentiate into the desired cell type(s). ← Survive in the recipient after transplant. ← Integrate into the surrounding tissue after transplant. ← Function appropriately for the duration of the recipient's life. ← Avoid harming the recipient in any way.

Also, to avoid the problem of immune rejection, scientists are experimenting with different research strategies to generate tissues that will not be rejected.

To summarize, stem cells offer exciting promise for future therapies, but significant technical hurdles remain that will only be overcome through years of intensive research.

VIII. Where can I get more information?

For a more detailed discussion of stem cells, see the NIH's Stem Cell Reports. Check the Frequently Asked Questions page for quick answers to specific queries. The navigation table at right can connect you to the information you need.

The following websites, which are not part of the NIH Stem Cell Information site, also contain information about stem cells. The NIH is not responsible for the content of these sites.

← http://www.isscr.org/public Stem cell information for the public from the International Society for Stem Cell Research (ISSCR).

← http://www.nlm.nih.gov/medlineplus/stemcells.html Medline Plus is a consumer health database that includes news, health resources, clinical trials, and more

← http://www.explorestemcells.co.uk A United Kingdom-based resource for the general public that discusses the use of stem cells in medical treatments and therapies.

← http://www.stemcellresearchnews.com A commercial, online newsletter that features stories about stem cells of all types.

Stem cell culture

Stem cells are immature cells which have the potential to grow into any kinds of cells/tissues, i.e skin, cardiac cells, blood cells, nerve cells etc.

Characteristic features of stem cells

1. Stem cells can replenish their numbers for long periods'through cell division.

Stem cells are immature cells which have the potential to grow into any kinds of cells/tissues, i.e skin, cardiac cells, blood cells, nerve cells etc.

Characteristic features of stem cells

1. Stem cells can replenish their numbers for long periods'through cell division.

2. These cells after receiving certain chemical signals can differentiate into specialised cells with specific functions.

3. Stem cells can be preserved for any number of years in liquid nitrogen at - 196° degree Celsius and can be used again when required.

Types of stem cells

There are mainly two types of stem 'cells : (i) Embryonic stem cells obtained from the inner mass of the developing blastocysts and (ii) Adult stem cells extracted from bone marrow.

(i) Embryonic stem cells : These are founfl in the early embryonic stages of vertebrates. The zygote formed when the sperm and egg unite is totipotent - that is, it

has the potential to give rise to the body in totality. The first few cell divisions in embryo development produce more totipotent cells. After four days of embryonic cell division, the cells begins to specialize. Then that embryo differentiate into inner cell mass made up of embryonic stem cells covered by one layered wall (Fig. 2.6.2). The inner cells are pluripotent, give rise to the progenitors* of cell lines such as blood cells, nerve cells, muscle cells etc. Prof. Alan Trounson's team (2004) at Monash University, Australia was the first in the world to create mature nerve cells from human embryonic stem cells. The team also announced that they have successfully transplanted nerve cells into the brain of a new born mice. The cells seemed to function like normal brain cells.

(ii) Adult stem cells : In the adults most cells are specialized with the exception of bone marrow stem cells and umbical cord blood stem cells. These stem cells can help in repair system, divide regularly to produce new specialized cells which may die or are lost.

Now with the advancement in technology, pluripotent stem cells have been more easily manipulated into specialized cells. The embryonic stem cells can be maintained and multiplied in vitro in the culture medium containing nutrients and leukaemia inhibitory factor. The pluripotent haematopoietic stem cell that is produced in the bone marrow and umbical cord has the ability to become an erythrocyte (red blood cells), a neutrophil, a basophil, a lympocyte, a monocyte, and a macrophage or mast cell. In vitro culture of stem cells also permit various manipulations for gene transfer. The cultured pluripotent embryonic stem cells are transfected with the appropriate transgene. Transfected ES cells are identified, selected and cloned.

Now scientists believe that they can program embryonic stem cells and those cells could be extracted and used to produce selected tissue or an entire organ for auto-transplant into the person who had supplied DNA or bone narrow cells. Since the tissues or organs would be genetically identical with the recipient there would be no fear of rejection and one could do away with the lifelong need to take immuno-suppressive drugs as recipients of donor organs now need to.

Applications of stem cell culture

1. The pluripotent stem cells have different therapeutic uses - i. It is used in the treatment of blood cancer. Chemotherapy is used to destroy all the bone marrow cells in the patient prior to transplants of bone marrow from healthy donors. The small volume of transplanted bone marrow eventually give rise to enough cells to take care of the body's need for blood forever.

ii. Dr. Karl Skorecki's team at Technion (Israel) has successfully turned human embryonic stem cells into insulin producing cells.

iii. The successful transfer of nerve cells derived from ES cells into the brain of new born mice seemed to function like normal brain cells. Such an approach shows real promise for treating neurodegenerative disease like Parkinson's disease.

2. Stem cells today are being used in heart disorders, neurological disorders like stroke, cerebral palsy (partial paralysis), liver regeneration, eye injuries etc. Dr. Carlos Lima (2006), a neurosurgeon of Portugal treated paralysed patients with stem cells. Some of the patients have been able to work, something which was impossible before. In India stem cells are being used in paralysed patients, diabetics and in patients with heart problems.

3. The ES cell culture could be used to produce embryos and harvest stem cells that could be used to repair damaged defective tissue.

4. Embryonic stem cell therapy is a future hope in organ transplants of human without immuno-suppressive drugs.

5. Embryonic stem cell culture could be used to repair damaged or defective tissue in the donor of sperms and eggs.

6. Embryonic stem cell culture technology is used to produce transgenic mice.

7. The haematopoetic umbical cord blood stem cell have been used to treat a number of blood an immune-system related genetic diseases and cancer.

Related tags

Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency

Rebecca J. McMurray ,1

Nikolaj Gadegaard ,2

P. Monica Tsimbouri ,1

Karl V. Burgess ,3

Laura E. McNamara ,1

Rahul Tare ,4

Kate Murawski ,4

Emmajayne Kingham ,4

Richard O. C. Oreffo 4 , 5

& Matthew J. Dalby 1

There is currently an unmet need for the supply of autologous, patient-specific stem cells for regenerative therapies in

the clinic. Mesenchymal stem cell differentiation can be driven by the material/cell interface suggesting a unique

strategy to manipulate stem cells in the absence of complex soluble chemistries or cellular reprogramming. However, so

far the derivation and identification of surfaces that allow retention of multipotency of this key regenerative cell type have

remained elusive. Adult stem cells spontaneously differentiate in culture, resulting in a rapid diminution of the

multipotent cell population and their regenerative capacity. Here we identify a nanostructured surface that retains stem-

cell phenotype and maintains stem-cell growth over eight weeks. Furthermore, the study implicates a role for small

RNAs in repressing key cell signalling and metabolomic pathways, demonstrating the potential of surfaces as non-

invasive tools with which to address the stem cell niche.

Subject terms:

Biological materials

Biomedical materials

Nanoscale materials

Figures at a glance

left

1. Figure 1: Expression of progenitor and osteoblast markers by MSCs cultured on SQ and on controls after four

weeks and eight weeks of culture.

a, On the planar control (flat) a heterogeneous cell population was formed that retained some level of STRO-1 and

ALCAM (MSC marker) expression, but also expressed osteocalcin (OCN) and osteopontin (OPN) (osteoblast marker). It

is noted that most of the cells had a fibroblast-like morphology. On the osteogenic controls, OCN and OPN expression

was noted and some level of the less specific progenitor marker, ALCAM, was retained—only low levels of STRO-1

were noted on NSQ50. On SQ, however, STRO-1 and ALCAM were still highly expressed by most cells, whereas no

expression of OCN and OPN was noted. It is noted that the cells had grown to confluence on the SQ surface. b, High

levels of ALCAM and STRO-1 were seen in MSCs cultured on SQ compared with cells cultured on the osteogenic

NSQ50. It is noted that STRO-1 (and CD63) is a more stringent marker for MSCs than ALCAM, which is expressed by

both stem cells and progenitor cells. That ALCAM is still expressed, but not STRO-1, on NSQ50 at eight weeks is

indicative that there are still osteoprogenitor cells present but that actual MSC numbers have dwindled. In all images,

green = phenotypic marker as indicated by the images, red = actin to give cell morphology, blue = nucleus; SEM images

of the SQ and NSQ50 surfaces are inset. c,d, Quantitative, real time (q)PCR at four weeks for progenitor markers

ALCAM (c) and CD63 (d) showed a statistically significant increase in expression between SQ and planar control, SQ

and NSQ50, and SQ and OGM. Graphs show mean±s.d., comparison by ANOVA— *p<0.05, **p<0.01, ***p<0.001, n=3.

Note: markings above the error bars denote comparison to flat control; comparison between SQ and NSQ50 is denoted

by a line.

2. Figure 2: MSC multipotency following prolonged culture on the SQ topography.

a, After 28 days of culture on SQ, cells were trypsinized, disaggregated and re-cultured on glass coverslips at 1×104 

cells ml−1. The cells were then cultured with adipogenic or osteogenic media for 24 and 72 h before immunostaining for

RUNX2 (runt-related protein 2, a transcription factor involved in osteogenesis) or PPARG (peroxisome proliferator-

activated receptor gamma, a transcription factor involved in adipogenesis). After 24 h, cells could be seen to express the

adipogenic and osteogenic transcription factors and this was intensified after 72 h. b, After 28 days of culture on planar

control or SQ, the MSCs were trypsinized and seeded onto glass coverslips at 1×104 cells ml−1 with adipogenic or

osteogenic media for 14 days before fixation and staining for the adipocyte marker PPARG or the osteoblast marker

osteopontin (OPN). After culture on SQ and when treated with adipogenic media, the cells could be seen to express

PPARG and had developed fat droplets (left image and magnification of the highlighted square). When treated with

osteogenic media, areas of dense, positive OPN expression were noted. However, after replating to coverslips post-

culture on planar control, levels of differentiation induction were much lower, with only small areas of PPARG or OPN

expression noted (arrows). c, After 28 days of culture on SQ, adipogenic or osteogenic media was added in situ with the

MSCs still on the SQ pattern and the cells cultured for a further 14 days before staining for PPARG or OPN. In response

to adipogenic media, the MSCs clustered and localized expression of PPARG was observed, although not to the same

extent as when disaggregated. In response to osteogenic media, the MSCs formed a limited number of osteoid clusters

positive for OPN. Blue=nucleus, red=actin, green=PPARG, Runx2 or OPN.

3. Figure 3: Pathway analysis of MSCs cultured on SQ, NSQ50 and with OGM compared with planar control.

a, Metabolic pathways after seven days of culture. MSCs on NSQ50 undergoing osteogenesis increased the expression

of transcripts relating to a large number of metabolic pathways. However MSCs on SQ down-regulated transcripts

corresponding to similar metabolic activity. b, At day seven, MSCs on SQ showed large-scale changes (mainly down-

regulation) of major canonical pathways. Oct 4, Nanog and pluripotency pathways were not significantly effected.

Although the NSQ50 initiated a range of metabolic pathways, it showed little change in canonical signalling compared to

control; OGM showed a number of changes (mainly up-regulations) in canonical signalling. c, At day 14 of culture,

MSCs on NSQ50 and with OGM showed a broad number of large functional changes (mainly up-regulations). MSCs on

SQ, however, showed very few changes (mainly down-regulations). Note that the threshold is significant at p<0.05 by

Fischer’s exact test and that the graph shows significance only as–log (p value) to show the significance as positive

values over a sensible scale. The up- and down-regulation data is shown in Supplementary Fig. S7. PPAR, peroxisome

proliferator-activated receptor; FGF, fibroblast growth factor; VEGF, vascular endothelial growth factor; MAPK, mitogen

activated protein kinase; PI3K/AKT, phosphoinositide 3-kinase; Oct 4, octamer-binding transcription factor 4; ERK5,

extracellular signal-regulated kinase 5; BMP, bone morphogenic protein; FAK, focal adhesion kinase; Cdc42, cell

division control protein 42 homolog.

4. Figure 4: Metabolic saturation in undifferentiated and differentiating MSCs.

Ratios were calculated for each metabolite, comparing metabolite abundance in MSCs cultured on the SQ, retention

and nanotopography relative to MSCs cultured in osteogenic differentiation media. Using the Kyoto Encyclopedia of

Genes and Genomes (KEGG) identification for each metabolite, the number of C=C bonds was identified. The number

of C=C bonds observed in undifferentiated cells was higher than in the differentiating cells.

5. Figure 5: MSC phenotype retention is linked to SNORD up-regulation.

Microarray-based SNORD profiling showing differential expressions in MSCs cultured on SQ, NSQ50 and treated with

OGM compared with MSCs cultured on planar control. For these 36 SNORDs, significant up-regulations (in all cases

p<0.01) in MSCs on SQ are plotted against significant down-regulations (in all cases p<0.05) in MSCs on NSQ50. The

cells treated with OGM follow the NSQ50 pattern more closely, as all the up-regulations in these cells are non-

significant. (n=3, bars=mean expression, comparison by ANOVA).

6. Figure 6: MSC phenotype retention is linked to intraceullular tension and ERK signalling.

a–c, Inhibition studies for actin/myosin contraction (a, RhoA kinase (ROCK) inhibition and b, blebbistatin) and

extracellular-signal related kinase (ERK) (c) in MSCs cultured on planar control, test topographies (SQ retention surface

and NSQ50 osteogenic surface) and with osteogenic media (OGM) for 14 days. When actin/myosin interaction was

blocked, expression of STRO-1 and osteopontin (OPN) dropped dramatically. However, PPARG expression was noted

as the low-tension default (arrows) (a,b). When ERK was inhibited neither STRO-1 expression nor functional

differentiation was noted, suggesting that both multipotency and differentiation are biochemically active states (c).

right

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Stem cell culture

Glycosan offers three options for Stem Cell Culture:

← HyStem™: for applications requiring attachment factor optimization. Extracellular matrix proteins can be mixed into the hydrogel and incorporated non-covalently before gelation. Alternatively, attachment peptides having an N-terminal cysteine can also be covalently linked to the matrix.

← HyStem-C™: a general starting point for optimization of a cell's microenvironment. HyStem-C contains Gelin-S or thiolated gelatin (denatured collagen) which allows attachment of a wide variety of cell types and takes the guesswork out of the appropriate attachment factors to use.

← HyStem-HP™: for applications requiring slow release of growth factors in a cell's microenvironment. HyStem-HP contains small amounts of thiolated heparin which ionically binds a wide variety of growth factors and slowly releases them over time17,18,19.

 

"HA hydrogels act as a unique microenvironment for the propagation of human embryonic stem cells (hESCs), likely due to the regulatory role of HA in the maintenance of hESCs in the undifferentiated state, in vitro as well as in vivo."3

HyStem™ hydrogels are designed for stem cell culture. They are chemically defined and wholly animal free.  They are based on

hyaluronan (HA) – a component of the extracellular matrix (ECM) that is abundant in embryos and stem cell niches. HyStem

hydrogels provide a compliant, viscoelastic matrix that is physiologically relevant for stem cells. They emulate a stripped down

version of the ECM to which additional components (such as growth factors and ECM proteins) can be added to recreate the

composition of a specific niche. 

Hyaluronan relevancy for stem cells

HyStem hydrogels are composed of HyStem (thiol-modified hyaluronan, HA) and Extralink™ (thiol-reactive crosslinking agent). HA

is the simplest glycosaminoglycan (a class of negatively charged polysaccharides) and a major constituent of the ECM1,2. Embryonic

ECM possesses a high quantity of glycosaminoglycans of which HA is predominant3. Human embryonic stem cells (H1, H9 and H13

lines) have been shown to express both CD44 and CD168 (RHAMM), which are HA receptors. Human embryonic stem cells (hESCs)

have also been cultivated by encapsulating them in HA hydrogels. These cells were grown for 15 days without detectable

differentiation. After recovery from the hydrogels, the hESCs could be differentiated using endothelial growth media supplemented

with VEGF3.

HA is also present in large amounts in the ECM of embryonic livers, fetal livers and the putative stem cell niche within the liver (the

Canals of Hering). Hepatic stem and progenitors cells (hepatoblasts) have been successfully cultured for over 4 weeks without

differentiation when encapsulated in HA hydrogels and grown with a defined media (Kubota’s medium). Additionally, these hepatic

progenitor cells express CD44 at high levels4.

Defined and animal-free hydrogel

When used with defined media, HyStem allows for the culture of stem cells in a defined system. The HA used to produce HyStem

is made by a proprietary bacterial fermentation process using the recombinant hasA gene from Streptococcus equisimilis,

which is expressed in Bacillus subtilis5, as the host in an ISO 9001:2000 process. It is 100% free of animal-derived raw

materials and no animal derived ingredients are used in its production. The polyethylene glycol diacrylate that is our Extralink is

an acrylate modification of polyethylene glycol (PEG), which is a commodity chemical . PEG is derived from petroleum and

inorganic sources and contains no animal source materials.

HyStem use

Encapsulation

Stem cells can be encapsulated in HyStem prior to crosslinking7, where they grow within the hydrogel matrix. Cells are removed from

the hydrogel by digesting it using hyaluronidase.

Surface growth

If ECM proteins are added to HyStem prior to crosslinking they are non-covalently incorporated into the matrix . If the relevant protein

is added, the stem cells can be plated on top of the hydrogel for pseudo three dimensional growth 1. Since the researcher determines

which ECM proteins to incorporate, the sourcing and concentration of the proteins is fully under their control. It may be optimal to

begin experimentation using animal derived ECMs that are compatible with humans, but less expensive. However, the method of

purification can affect its performance as can the specific isoform of the ECM protein. Therefore, results achieved with animal

proteins will not necessarily be mirrored in their commercially available human counterparts.

Additionally, once the relevant ECM proteins are determined for your cell type, we recommend doing a concentration variation study

to determine the minimum protein amount required for your application. Cells are passaged using dispase or collagenase.

HyStem hydrogel variation

ECM protein incorporation

Stem cells expand when encapsulated in HA only hydrogels. However, of the cells tested to date, none attach to HyStem

hydrogels1,2. For most applications, optimal cell proliferation will require attachment. This can be achieved by incorporating specific

ECM proteins into the HyStem™ hydrogels prior to crosslinking. Since the appropriate ECM protein depends upon the cell type and

the desired outcome (proliferation without differentiation vs differentiation), HyStem™ gives the user the flexibility to decide on the

appropriate experimental conditions and material sources (human vs animal).

During embryogenesis, laminin is the first ECM protein to be excreted within the basement membrane. It is observed in a punctuate

pattern in the intercellular spaces between cells in the 8 cell stage embryo. Later in development fibronectin, heparan sulfate and

collagen IV accumulate in the same area. The deposition and self-assembly of collagen IV leads to the organization of the basement

membrane and hence to the organization of the attached cells, leading to the polarization of the epithelial monolayer8.

Rigidity

The rigidity of HyStem hydrogels can easily be adjusted. Depending upon your application, the hydrogel stiffness has been found to

be very important in stem cell cultures9,10. The standard HyStem hydrogel made per our instructions has a compliance of ~300 Pa11.

The hydrogel compliance can also be altered either by varying the amount of crosslinker or by diluting the hydrogel solutions.

Growth factor incorporation

HyStem may be used to conserve growth factors (GFs) while extending their active life.   For media that use GFs, it is possible to

move the GFs from the media to the HyStem™ hydrogel. GFs are retained within and slowly released from the HyStem-based

hydrogels over several weeks12,13. They are protected from proteolysis so that their bioactivity is maintained12,13. To date we have

characterized release of 7 GFs (bFGF, VEGF, KGF, PDGF, TGF-β1, Ang-1, HGF)12,13,14. Experimentation will be required to

determine the optimal GF concentration in the hydrogel for your application.

Stem cells cultured

The following stem cells have been cultured in HyStem-based hydrogels:       

← human embryonic stem cells (H9)7

← human cord blood derived CD34+ stem cells7

← human mesenchymal stem cells7

← human hepatic progenitor cells4

← rabbit mesenchymal stem cells15

References

1. X.Z. Shu, S. Ahmad, Y. Liu, and G.D. Prestwich, “Synthesis and Evaluation of Injectable, in situ Crosslinkable Synthetic

Extracellular Matrices (sECMs) for  Tissue Engineering,” J. Biomed Mater. Res. A, 79A(4), 901-912 (2006).

2. X.Z. Shu, Y. Liu, F. Palumbo, G.D. Prestwich, “Disulfide-crosslinked Hyaluronan-Gelatin Hydrogel Films: A Covalent Mimic

of the Extracellular Matrix for In Vitro Cell Growth,” Biomaterials, 24, 3825-3834 (2003).

3. S. Gerecht, J.A. Brudick, L.S. Ferreira, S.A. Townsend, R. Langer, G. Vunjak-Novakovic, “Hyaluronic acid hydrogel for

controlled self-renewal and differentiation of human embryonic stem cells,” PNAs 104(27): 11298-11303.

4. Unpublished data from W. Turner, R. Turner, L. Reid, University of North Carolina (publications submitted August 2007).

5. B.Widner, R. Behr, S. Von Dollen, M. Tang, T. Heu, A. Sloma, D. Sternberg, P.L. DeAngelis, P.H. Weigel, S. Brown,

“Hyaluronic Acid Production in Bacillus subtilis”, Applied and Environmental Microbiology 71(7): 3747-3752 (2005).

6. S. Cai, Y. Liu, X.Z.Shu, G.D.Prestwich, “Injectable glycosaminoglycan hydrogels for controlled release of human basic

fibroblast growth factor,” Biomaterials, 26, 6054-6067 (2005).

7. Unpublished data from T. Tandenski, L. Kelley, University of Utah.

8. Ingber, D. “Mechanical control of tissue morphogenesis during embryological development,” Int. J. Dev. Biol. 50: 255-266

(2006).

9. A.J. Engler. S. Sen. H.L. Sweeney, D.E. Discher, ”Matrix Elasticity Directs Stem Cell Lineage Specification,” Cell, 126, 677-

689 (2006).

10. T. Yeung, P.C. Georges, L.A. Flanagan, B. Marg, M. Ortiz, M. Funaki, N. Zahir, W. Ming, V. Weaver, P.A. Jamney, “Effects

of Substrate Stiffness on Cell Morphology, Cytoskeletal Structure, and Adhesion, “Cell Motility and the Cytoskelton", 60, 24-

34 (2005).

11. Unpublished data from Janssen Vanderhooft, Glenn Prestwich, University of Utah.

12. D. B. Pike, S. Cai, K.R. Pomraning, M.A. Firpo, R.J. Fisher, X.Z. Shu, G.D. Prestwich, R.A. Peattie, “Heparin-regulated

release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and

bFGF”, Biomaterials, 27, 5242–5251 (2006).

13. Unpublished data from Rob Peattie lab (Oregon State University) and S. Cai and B. Yu of Glenn Prestwich (University of

Utah) lab.

14. Unpublished data from Yongzhi Qiu, Robert McCall, Vladimir Mironov, Xuejun Wen, Clemson University and Medical

University of South Carolina.

15. Y. Liu, X.Z. Shu, G. D. Prestwich, “Osteochondral defect repair with autologous bone marrow derived MSC cells in an

injectable in situ crosslinked synthetic extracellular matrix” Tissue Engineering, Tissue Eng., 12(12),  3405-3416 (2006).

16. X. Z. Shu, K. Ghosh, Y. Liu, F. S. Palumbo, Y. Luo, R. A. Clark, and G. D. Prestwich, "Attachment and spreading of

fibroblasts on an RGD peptide-modified iInjectable hyaluronan hydrogel" J. Biomed. Mat. Res. 68A, 365-375 (2004).

17. S. Cai, Y. Liu, X.Z. Shu, G.D. Prestwich, "Injectable glycosaminoglycan hydrogels for controlled release of human basic

fibroblast growth factor", Biomaterials, 26, 6054-6067 (2005).

18. D. B. Pike, S. Cai, K.R. Pomraning, M. A. Firpo, R. J. Fisher, X. Z. Shu, G. D. Prestwich, R. A. Peattie, "Heparin-regulated

release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and

bFGF", Biomaterials, 27, 5242-5251 (2006).

19. Unpublished data from G. D. Prestwich, et al, University of Utah, and R. Peattie, et al, University of Oregon.

Culture of Human Embryonic Stem Cells (hESC)

All cell lines are initially grown according to the supplier's protocols but we are adapting them to one simple protocol outlined below:

← 6-well plates (Falcon Cat #353046) are coated for 20 to 60 minutes at room temperature with 0.1% gelatin (Sigma Cat #G1890) in dH2O.

← Mouse embryonic fibroblasts (CF1 strain), cultured in MEF medium, are mitotically inactivated by treatment with 10μg/ml mitomycin C (Roche Cat #107 409) for 2 to 3 hours at 37°C. Cells are washed three to four times with PBS, trypsinized (Invitrogen Cat #25300-054), and plated at a density of 0.75 x 105/ml with 2.5ml per well of a gelatin-coated 6-well dish. Alternatively, cells may be inactivated by exposure to 8000rads of X-irradiation and plated at the same density.

← Immediately before plating hESC, MEFs are rinsed once or twice with PBS. hESC are plated onto MEFs as small clumps in 2.5ml per well of hESC medium containing 4ng/ml bFGF (R&D Systems Cat #233-FB). Cells are fed every day until ready to passage which is determined by the size of colonies, the age of MEFs (should not be older than 2 weeks) or differentiation status of the cells.

← Colonies which appear to be differentiating, are manually removed before passaging. ← To passage hESC, cells are washed once or twice with PBS and incubated with filter-sterilized 1mg/ml collagenase IV

(Invitrogen Cat #17104-019) in DMEM/F12 for 10 to 30 minutes. Plates should be agitated every 10 minutes until colonies begin to detach. When moderate tapping of the plate causes the colonies to dislodge, they are collected and the wells washed with hESC medium to collect any remaining hESC. Alternatively, colonies may be removed using a cell scraper and collected.

← Colonies are allowed to sediment for 5 to 10 minutes. The supernatant, containing residual MEFs, is aspirated, and the colonies are washed with 5ml hESC medium and allowed to sediment again. This is repeated once more.

← After the final sedimentation, the colonies are resuspended in 1ml of hES medium and triturated gently to break up the colonies to approximately 100-cell size. Generally, cell lines are passaged at a ratio of between 1:3 and 1:6 every four to seven days.

MEF Medium

DMEM Invitrogen 11965-092 450ml

Heat-inactivated FBS Invitrogen 16000-044 50ml

Non-essential amino acids Invitrogen 11140-050 5ml

L-Glutamine Invitrogen 25030-081 5ml

hESC Medium

DMEM/F12 Invitrogen 11330-032 400ml

Knockout Serum Replacer Invitrogen 10828-028 100ml

Non-essential amino acids Invitrogen 11140-050 5ml

L-Glutamine Invitrogen 25030-081 2.5ml

β-mercaptoethanol Sigma 7522 3.5μl

17 July 2011 Last updated at 17:00 GMT

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Scientists find 'better way' to grow adult stem cells

A stem cell makes adhesions (shown in green) to the new "nano-patterned" plastic surface Continue reading the main story

Related Stories

Stem cell hope for heart patients First synthetic organ transplant Parkinson's artificial brain bank

A new plastic surface which overcomes the difficulties associated with growing adult stem cells has been developed, according to scientists.

Standard surfaces have proved limited for growing large amounts and retaining the stem cells' useful characteristics.

It is hoped the discovery could lead to the creation of stem cell therapies for re-growing bone and tissue, and also for conditions such as arthritis.

The study was carried out by Glasgow and Southampton universities.

The new "nano-patterned" surface was created using a manufacturing process similar to that used to make Blu-ray discs.

The surface is covered with tiny pits, which the researchers said made it more effective in allowing stem cells to grow and spread into useful cells for therapy.

Currently, when adult stem cells are harvested from a patient, they are then cultured in a laboratory to increase the quantities of cells and create a batch of sufficient volume to kick-start the process of cellular regeneration.

Continue reading the main story

“Start Quote

The implications for research and future interventions for patients with arthritis and other musculoskeletal diseases are substantial”

End Quote Prof Richard Oreffo University of Southampton

At this point they can be reintroduced back into the patient.

The process of culturing is made difficult because stem cells grown on standard plastic tissue culture surfaces do not always expand to create new stem cells but instead create other cells which are of no use in therapy.

Stem cell expansion can be boosted by immersing the cells in chemical solutions, but the scientists said these methods were limited in their effectiveness.

Dr Matthew Dalby, from the University of Glasgow, led the research alongside colleague Dr Nikolaj Gadegaard and Prof Richard Oreffo of the University of Southampton.

'Stem cell factories'

Mr Dalby said: "This new nano-structured surface can be used to very effectively culture mesencyhmal stem cells, taken from sources such as bone marrow, which can then be put to use in musculoskeletal, orthopaedic and connective tissues.

"If the same process can be used to culture other types of stem cells too - and this research is under way in our labs - our technology could be the first step on the road to developing large-scale stem cell culture factories, which would allow for the creation of a wide range of therapies for many common diseases such as diabetes, arthritis, Alzheimer's disease and Parkinson's disease."

The surface is covered with tiny pits, which help stem cells to grow and spread

He said the group hoped to make the surface commercially available.

Prof Oreffo added: "It is important to realise the ability to retain skeletal stem cell phenotype using surface topography offers a step change in current approaches for stem cell biology.

"The implications for research and future interventions for patients with arthritis and other musculoskeletal diseases are substantial."

The study was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and the University of Glasgow.

The paper, Nanoscale surfaces for the long-term maintenance of mesenchymalstem cell phenotype and multipotency, was published in the journal Nature Materials.

8 July 2011 Last updated at 08:43 GMT

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Stem cell hope for heart patients

Angina can be debilitating Continue reading the main story

Related Stories

Morning heart attacks 'are worse' 'Leftover' veins yield stem cells

Scientists have raised hope that stem cell therapy could provide significant relief for patients disabled by untreatable chest pain.

Patients with severe angina had stem cells from their blood injected into their heart.

The therapy, carried out by Chicago's Northwestern University, halved the number of bouts of angina chest pain.

But UK experts have stressed the work is still at an early stage, and the potential longer benefit is unknown.

The procedure may also carry a risk: it is suspected of causing heart muscle damage in two patients, and others reported bone and chest pain.

The study, reported in the journal Circulation Research, was carried out on 167 patients with "refraction" angina, which does not respond to any standard treatment.

They were given high or low dose stem cell infusions, or a dummy injection.

A year on, patients in the low-dose group had an average of 6.3 episodes of pain a week, compared to 11 a week for those given the placebo jab.

Continue reading the main story

“Start Quote

It translates as going from being able to walk slowly to being able to ride a bike.”

End Quote Professor Douglas Losordo Northwestern University

The length of time they were able to tolerate exercise also improved by 139 seconds after six months, compared to an improvement of 69 seconds for the placebo group.

There was no significant benefit from receiving a higher dose of stem cells.

Lead researcher Professor Douglas Losordo said: "The net difference in exercise tolerance is highly clinically significant, particularly in a patient population that is severely limited by symptoms.

"It translates as going from being able to watch television to being able to walk at a normal pace or going from being able to walk slowly to being able to ride a bike."

Bone marrow cells

The treatment used bone marrow stem cells called CD34+ cells which circulate in the blood.

Previous research has suggested these cells can create new blood vessels in diseased heart muscle.

The researchers used a growth-stimulated drug to boost their numbers before harvesting them.

The cells were then injected into areas of heart muscle that had been starved of blood.

The Chicago team plans to further develop the technique in more advanced Phase III trials later this year.

Professor Jeremy Pearson, associate medical director at the British Heart Foundation, said the study showed promise, but warned it was still uncertain whether the therapy would produce lasting benefit.

He said: "The mechanisms involved are still poorly understood.

"Until these uncertainties are resolved, it remains unclear how successful this kind of treatment will prove to be."

Surgeons carry out first synthetic windpipe transplant By Michelle Roberts Health reporter, BBC News, in Stockholm

The replacement windpipe was grown in the lab Continue reading the main story

Related Stories

Synthetic transplant surgery Windpipe transplant breakthrough Stem cell windpipe op 'success'

Surgeons in Sweden have carried out the world's first synthetic organ transplant.

Scientists in London created an artificial windpipe which was then coated in stem cells from the patient.

Crucially, the technique does not need a donor, and there is no risk of the organ being rejected. The surgeons stress a windpipe can also be made within days.

The 36-year-old cancer patient is doing well a month after the operation.

Professor Paolo Macchiarini from Italy led the pioneering surgery, which took place at the Karolinska University Hospital.

In an interview with the BBC, he said he now hopes to use the technique to treat a nine-month-old child in Korea who was born with a malformed windpipe or trachea.

Professor Macchiarini already has 10 other windpipe transplants under his belt - most notably the world's first tissue-engineered tracheal transplant in 2008 on 30-year-old Spanish woman Claudia Costillo - but all required a donor.

Indistinguishable

The key to the latest technique is modelling a structure or scaffold that is an exact replica of the patient's own windpipe, removing the need for a donor organ.

To do this he enlisted the help of UK experts who were given 3D scans of the 36-year-old African patient, Andemariam Teklesenbet Beyene. The geology student currently lives in Iceland where he is studying for a PhD.

Using these images, the scientists at University College London were able to craft a perfect copy of Mr Beyene's trachea and two main bronchi out of glass.

This was then flown to Sweden and soaked in a solution of stem cells taken from the patient's bone marrow.

After two days, the millions of holes in the porous windpipe had been seeded with the patient's own tissue.

Dr Alex Seifalian and his team used this fragile structure to create a replacement for the patient, whose own windpipe was ravaged by an inoperable tumour.

Despite aggressive chemotherapy and radiotherapy, the cancer had grown to the size of a golf ball and was blocking his breathing. Without a transplant he would have died.

During a 12-hour operation Professor Macchiarini removed all of the tumour and the diseased windpipe and replaced it with the tailor-made replica.

The bone marrow cells and lining cells taken from his nose, which were also implanted during the operation, were able to divide and grow, turning the inert windpipe scaffold into an organ indistinguishable from a normal healthy one.

And, importantly, Mr Beyene's body will accept it as its own, meaning he will not need to take the strong anti-rejection drugs that other transplant patients have to.

Professor Macchiarini said this was the real breakthrough.

"Thanks to nanotechnology, this new branch of regenerative medicine, we are now able to produce a custom-made windpipe within two days or one week.

"This is a synthetic windpipe. The beauty of this is you can have it immediately. There is no delay. This technique does not rely on a human donation."

He said many other organs could be repaired or replaced in the same way.

A month on from his operation, Mr Beyene is still looking weak, but well.

Sitting up in his hospital bed, he said: "I was very scared, very scared about the operation. But it was live or die."

He says he is looking forward to getting back to Iceland to finish his studies and then returning to his home in Eritrea where he will be reunited with his wife and young family, and meet his new three-month-old child.

He says he is eternally grateful to the medical team that has saved his life.

Parkinson's artificial brain bank

By Pallab Ghosh Science correspondent, BBC News

Brain cells grown from the skin cells of a Parkinson's patient are likely to deteriorate Continue reading the main story

Related Stories

Skin cells 'turned into neurons' Parkinson's skin reprogrammed into stem cell

Researchers in Oxford have begun creating a bank of artificially grown brain cells from Parkinson's patients, BBC news has learned.

They are using a new stem cell technique that allows them to turn a small piece of skin from the patient into a small piece of brain.

This is the first time this has been done in a large-scale study aimed at finding cures for the disease.

Researchers say they can analyse nerve cells as they start to deteriorate.

The first batch of nerve cells have been grown from a 56-year-old Oxfordshire man, Derek Underwood.

He had to take early retirement because of the progression of the disease.

Mr Underwood will be the first of 50 patients whose skin cells will be grown into brain cells as part of a five year study.

According Dr Richard Wade Martins of Oxford University, who is leading the study, the aim is to build up a "brain bank" which will enable researchers to study how the disease develops in unprecedented detail.

"The brain is an inaccessible organ and you can't get bits of people's brain to study very easily," he said.

"But what we have here is a disease in a dish, that are just like Derek's brain cells but are accessible and can be produced in unlimited quantities"

Lab brain

The first step, according to Dr Michelle Hu of the John Radcliffe Hospital in Oxford, is to compare the brain cells grown from Parkinson's patients, with those grown from healthy volunteers and see how they differ.

"For the first time we can look at the cells before they deteriorate and look at the earliest changes," she said.

"We can look at what cellular processes are happening that make the cells die and learn why it is that the cells get sick. And we want to see if there are any treatments we can offer to reverse that process and help patients regain normal function."

This is the first large scale clinical study to use a technique which was developed by Japanese scientists three years ago, called "induced pluripotent stem cell" or IPS for short.

Genes are inserted into the skin cells, reprogramming them to become something else.

IPS is similar to the embryonic stem cell technique which was used to create Dolly the Sheep, but IPS does not result in the creation of an embryo and so is regarded by some as an ethically more acceptable approach.

15 June 2011 Last updated at 08:26 GMT

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Surgical research spurned for 'sexy' science projects

By Pallab Ghosh Science correspondent, BBC News

The UK is behind the US and many parts of Europe in using the latest surgical techniques

The Royal College of Surgeons says many patients are dying, or remaining ill for longer, because surgical research is being starved of funds.

In a new report the RSC has called for a larger slice of research funding.

The College estimates that surgery receives 1.5% of the £1.5bn that's currently spent on medical research.

The next President of the RSC, Norman Williams said funding bodies were instead backing fashionable research areas such as stem cells and genetics.

"Our academic institutions have concentrated on non-surgical research because surgery hasn't been sexy," he said.

Continue reading the main story

“Start Quote

Our academic institutions have concentrated on non-surgical research because surgery hasn't been sexy”

End Quote Professor Norman Williams President-elect Royal College of Surgeons

According to Professor Williams, the current system of testing new medicines and surgical techniques is flawed and discourages surgeons from developing and testing new methods.

This is reflected, he said, in the fact that just 11 surgical trials were financed by funding bodies in 2009.

"This is a major problem across the country. We are finding it extremely difficult to develop new technology, to train people to use that new technology and to use that to disseminate it to different centres to get it established".

The report calls for a new system to pilot and trial new techniques.

Surgery cures

It argues that a small investment in developing new techniques could make a big difference to patient care.

Professor Williams said his own field of bowel surgery has been revolutionised by a new surgical technique which has improved survival rates, far more than radiotherapy or chemotherapy.

Medical Research focussed on fashionable areas of research such as stem cells, according to RSC

Although the technique was developed in the UK, the surgeon who developed the technique could not get backing for clinical trials to be carried out in the UK so it was tested out in Scandinavia: a factor which delayed its routine use in the UK by 20 years," he said.

The report, also said surgeons themselves were partly to blame arguing the profession does not have a research culture and there is no system to spread best practice.

Not accurate

The situation could be improved, according to the RCS, if the NHS Commissioning Board (NCB) encouraged the spread of successful techniques. It also suggested that NHS Trusts should encourage and support trials.

In response to the report, the Medical Research Council said that it's "not accurate" to say it is not supportive of research in this area.

Dr Declan Mulkeen, who is the director of the MRC's research programmes, said: "We should be optimistic rather than pessimistic about how much surgical research has won funding over the past few years and what the potential is for it to be supported even more over the next while."

A Department of Health spokesperson said efforts were being made to encourage surgical research.

"We invest £20 million in a regional innovation fund each year to share the most innovative practices and treatments across the NHS. And over the next four years we have committed over £4 billion to high quality medical research - including surgical research.

"We encourage surgeons to apply for this funding as we want to support new ideas and research that meets NIHR requirements and helps us to deliver high quality care to patients."

Stem cell hope for osteoarthritis

Stem cells offer a potential

way to repair cartilage

damaged by osteoarthritis, say

scientists.

They have identified a type of

stem cell which can be

transformed into cartilage cells

known as chondrocytes.

In theory, it should be possible to

create new chondrocytes in

sufficient numbers to achieve a

real therapeutic effect for

osteoarthritis patients.

The Cardiff University work was

presented to the UK National

Stem Cell Network Annual Science

Meeting.

Osteoarthritis, which affects more

than two million people in the UK,

occurs when changes in the make-up of the body's cartilage causes

joints to fail to work properly.

At its worse it can bring about the break-up of cartilage, causing the

ends of the bones in the joint to rub against each other.

This results in severe pain and deformation of the joint.

One current treatment for younger patients is to harvest cartilage

cells from neighbouring healthy cartilage and transplant them into

The cells may repair cartilage (Copyright: Cardiff University)

If we can translate these

successes from the laboratory

into treating patients the

possibility opens up of making a

remarkable impact on this

common painful and disabling

condition

Professor Alan Silman

Arthritis Research Campaign

the damaged area. Unfortunately, only a limited number of cells can

be generated.

Immature stem cells have the ability to become any tissue in the

body.

However, the cell identified by the Cardiff team is at a more

advanced stage, where it has lost some of its plasticity but not its

ability to become a chondrocyte if cultured in the lab in the right

way.

Real benefits

Lead researcher Professor Charlie Archer said: "We have identified a

cell which, when grown in the lab, can produce enough of a person's

own cartilage that it could be effectively transplanted.

"There are limitations in trying to transplant a patient's existing

cartilage cells but, by culturing it from a resident stem cell, we

believe we can overcome this limitation.

"This research could have real benefits for arthritis sufferers and

especially younger active patients with cartilage lesions that can

progress to whole scale osteoarthritis."

The Cardiff team have now started animal tests, and hope to launch

a clinical trial next year.

Professor Alan Silman, medical director of the Arthritis Research

Campaign, which part-funded the study, said: "How to stop or even

reverse the wearing away of cartilage that is the hallmark of

osteoarthritis has been a treatment goal which up to now has not

proved possible.

"If we can translate these successes from the laboratory into treating

patients the possibility opens up of making a remarkable impact on

this common painful and disabling condition."

Cell Culture Media Supplements & Reagents

← Complete Listing of Cell Culture Media & Supplements

Culture Media and Supplements

Mouse Embryonic Fibroblast Conditioned Media (Catalog # AR005)

Serum-free media (80% Knockout™ D-MEM, 20% Knockout serum replacement, 1% MEM non-essential

amino acids, 2 mM GlutaMAX™, 0.1 mM beta-mercaptoethanol, 4 ng/mL FGF basic) was conditioned by

gamma-irradiated CF-1 fibroblasts at 37° C for 24 hours.

A B

SSEA-4 and Oct-3/4 Expression in Embryonic Stem Cells. Human embryonic stem cells were cultured with recombinant human FGF basic

(Catalog # 233-FB) in the presence (A) or absence (B) of Mouse Embryonic Fibroblast (MEF)-conditioned medium (Catalog # AR005). SSEA-4 and Oct-3/4 were detected using anti-human SSEA-4 monoclonal antibody (red, Catalog #

MAB1435) and anti-human Oct-3/4 polyclonal antibody (green, Catalog # AF1759). Cells were counterstained with DAPI (blue). Image courtesy of Dr.

Frank Soldner of the National Institutes of Health.

StemXVivo™ Culture Media

StemXVivo media products are specifically designed for stem/progenitor cell culture. All the components included within

these defined, basal, serum-free formulations have been selected, optimized, and validated for the specific cell type of

interest.

N-2 Plus Media Supplement

A serum-free, chemically defined, concentrated media supplement formulated to provide optimal growth conditions for

neural stem cell expansion (Catalog # AR003). N2 is composed of Bovine Insulin, Human Transferrin, Putrescine, Selenite,

and Progesterone. The supplement is supplied as a 100X concentrate in water.

Resazurin

Resazurin can be utilized as a simple and quantitative method for measuring cell proliferation, viability, and cytotoxicity. It

is a non-toxic, water soluble, redox-sensitive dye that changes from its blue/non-fluorescent state to a pink/highly

fluorescent state upon reduction by viable cells. Fluorescence can be read using 544 nm excitation and 590 nm emission

wavelengths or absorbance can be read using a spectrophotometer set at 570 nm.

← Label: Resazurin changes from blue/non-fluorescent to pink/highly fluorescent

← Testing Format: Microplate reader

← Sample Type: Cultured cells

← Size: 100 mL (Catalog # AR002)

Additional Reagents

Extracellular Matrix Molecules

R&D Systems offers a range of recombinant ECM molecules which can be applied to cell culture plates to promote cell

adhesion.

Basement Membrane Extracts

Basement Membrane Extracts promote and maintain differentiated phenotypes of cells in culture.

Cultrex Poly-L-Lysine

A highly positively charged amino acid chain commonly used as a coating agent to promote cell adhesion in culture

(Catalog # 3438-100-01).

Holo-Transferrin

Holo-Transferrin facilitates the delivery of cytosolic iron through a receptor-mediated process (Catalog # 2914-HT).

Polystyrene Culture Microplates

R&D Systems offers packs of high-binding, flat-bottom polystyrene 96-well microplates. Each microplate consists of twelve

removable strips of 8 wells and are available in both clear (Catalog # DY990) and black (Catalog # DY991).

Knockout, GlutaMAX, and KSR are trademarks of Life Technologies, Carlsbad, CA.GlobalStem ES-DMEM is a trademark of GlobalStem, Rockville, MD.

Extracellular Matrix Molecules

In addition to pre-coated microplates, R&D Systems offers a range of recombinant ECM molecules which can be applied to

cell culture plates to promote cell adhesion.

Many proteins of the ECM interact with cells via cell surface integrin family receptors. The resulting focal contacts are

important for the maintenance of tissue architecture and for supporting a variety of cellular processes. ECM

molecule/integrin binding may initiate a complex network of signal transduction cascades that, depending on the context,

play an important role in cell spreading, migration, proliferation, and differentiation during embryogenesis, wound healing,

and tumor development.

Agrin

A heparan sulfate proteoglycan, Agrin is a component of the synaptic basal lamina.

Aggrecan

Also known as Aggrecan-1, chondroitin sulfate proteoglycan, and large aggregating proteoglycan, Aggrecan is the key

component of the cartilage extracellular matrix.

Collagen

Type-I collagen is the major structural component of extracellular matrices found in connective tissue and internal organs.

It is most prevalent in the dermis, tendons, and bone.

Decorin

Decorin is a small secreted chondroitin/dermatan sulfate proteoglycan in the family of small leucine-rich proteoglycans

(SLRPs). Decorin has been implicated in matrix assembly.

Fibronectin

One of the primary cell adhesion molecules, Fibronectin plays an important role in normal morphogenesis, including cell

adhesion, migration, differentiation, and specific gene expression.

Laminin-1

This highly purified preparation of mouse Laminin-1 increases cell adhesion, migration, growth, and differentiation.

MEPE

Matrix Extracellular Phosphoglycoprotein (MEPE), also known as OF45 in mouse and rat, is primarily expressed in bone and

dentin.

Nidogen-1

Also know as entactin, Nidogen-1 is a secreted, monomeric glycoprotein that serves as a major linking component of

basement membranes.

F-Spondin

Also known as Spondin-1 and VSGP, F-Spondin is a secreted, heparan-binding extracellular matrix glycoprotein.

R-Spondin

R-Spondins (Roof plate-specific Spondin) are expressed in early development at the roof plate boundary and are thought to

contribute to dorsal neural tube development.

T enascin

Tenascins are high molecular weight extracellular matrix glycoproteins that have a complex spatial and temporal pattern of

expression during embryogenesis, wound healing and neoplastic processes.

Testican

Testicans are proteoglycans which are highly expressed in brain tissue.

Vitronectin

A larger glycoprotein involved in a number of biological functions including cell adhesion, cell spreading migration,

proliferation, extracellular anchoring, fibrinolysis, hemostasis, and complement immune defense.

R&D Systems also offers traditional synthetic products to promote cell adhesion.

Poly-L-Lysine

A highly positively charged amino acid chain, commonly used as a coating agent to promote cell adhesion in culture

(Catalog # 3438-100-01).

Culture Microplates

R&D Systems offers packs of high-binding, flat-bottom polystyrene 96-well microplates. Each microplate consists of twelve

removable strips of 8 wells and are available in both clear (Catalog # DY990) and black (Catalog # DY991).

Extracellular Matrix Pre-coated Plates

96-well microplates pre-coated with fibronectin or vitronectin.

Basement Membrane Extracts

An alternative approach to promote cell adhesion.

Basement Membrane Extracts

← Complete Listing of Basement Membrane Extracts

Basement Membrane Extracts promote and maintain differentiated phenotypes of cells in culture.

Basement membranes are continuous sheets of specialized extracellular matrix that form an interface between endothelial,

epithelial, muscle, or neuronal cells and their adjacent stroma. Cultrex® Basement Membrane Extract (BME) is a soluble

basement membrane extract of the Engelbreth-Holm-Swarm (EHS) tumor. All Cultrex Basement Membrane Extracts

undergo vigorous sterility testing with no bacteria or fungi detected after incubation at 37° C for 14 days. They are negative

for mycoplasma by PCR and the Endotoxin level is XX 20 EU/mL by LAL. These specialized solutions gel within 30 mins at

37° C to form a reconstituted basement membrane on any desired growth surface. Use BME to promote and maintain a

differentiated phenotype of cell cultures such as epithelial cells, endothelial cells, glandular cells, neurons, and smooth

muscle cells.

Product features

← Rapid - gels within 30 minutes at 37º C.

← Reliable -maintains gelled form in culture medium for 14 days.

← Sterile - screened for bacteria, fungi, and mycoplasma.

← Specific - available with reduced growth factor content, with or without Phenol Red, and additional sterility testing.

Cultrex Basement Membrane Extract

BME consists of laminin I, type IV collagen, entactin, and heparan sulfate proteoglycan.

Cultrex Basement Membrane Extract - without Phenol Red

Since Phenol Red has been shown to act as a differentiating factor and may affect some colorimetric or fluorescence-based

assays, extracts are available without Phenol Red.

Cultrex Basement Membrane Extract – Reduced Growth Factor

For studies focused on cell growth, BME is subjected to a special process to reduce growth factor content.

Growth Factor BME (standard) BME (reduced growth factor)

bovine FGF 0-0.1 pg/mL 0-0.1 pg/mL

human EGF 0.5-13 ng/mL < 0.5 ng/mL

human IGF-I 15.6 ng/mL 5.0 ng/mL

human PDGF 12 pg/mL < 5.0 pg/mL

human NGF < 0.2 ng/mL < 0.2 ng/mL

human TGF-beta 2.3 ng/mL 1.7 ng/mL

Cultrex Basement Membrane Extract – PathClear®

PathClear products undergo additional testing and are confirmed negative by PCR for 31 infectious organisms and viruses,

including the LDEV virus.

Cultrex 3-D Culture Matrix™

Specifically produced for and qualified in 3-D culture studies.

Cultrex Basement Membrane Extract consists of laminin I, type IV collagen, entactin, and heparan sulfate proteoglycan.

Basement Membrane Extracts specific for individual basement membrane proteins of interest are also available.

Basement Membrane-Specific Extracts

← Mouse Laminin I (Catalog # 3400-010-01)

← Bovine Collagen I (Catalog # 3442-050-01)

← Rat Collagen I (Catalog # 3440-100-01)

← Mouse Collagen IV (Catalog # 3410-010-01)

← Cultrex Cell Staining Kit (Catalog # 3437-100-K)

The Cell Staining solution contains a mixture of Azur A and Methylene Blue, specially formulated to provide optimized

staining of cells and structures grown on Basement Matrix Extract.

BME Cell Invasion Assays

Fluorescence-based 96-well assays for investigating chemotaxis, cell migration, and cell invasion.

Cultrex and PathClear are registered trademarks of Trevigen, Inc.3-D Culture Matrix is a trademark of Trevigen, Inc.

Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are bone marrow-derived, self-renewing, and

multipotent progenitors. MSCs have been shown to be capable of

differentiating into multiple cell types including adipocytes, chondrocytes,

osteocytes, and cardiomyocytes. R&D Systems offers kits for the maintenance

and expansion of MSCs, as well as kits and reagents designed to promote and

identify the progression of MSCs into osteogenic, adipogenic, and chondrogenic lineages.

A Summary of Mesenchymal Stem Cell Molecules

Media and Supplements for Mesenchymal Stem Cells

Culture media specifically optimized for use with mesenchymal stem cells.

Mesenchymal Stem Cell Identification Kits

Kits contain specially formulated media supplements and a panel of antibodies for the differentiation and identification of

osteocytes, adipocytes, and chondrocytes.

Marker Panels for Mesenchymal Stem Cells

Antibody panels to assess differentiation status and identify specific cell types of interest.

Multi-Color Flow Cytometry Kits for Mesenchymal Stem Cells

Kits to simultaneously label cells using CD105-PerCP, CD146-Fluorescein, CD90-APC, and CD45-PE.

StemXVivo™ Culture Matrix

A defined proprietary mixture of recombinant human adhesion molecules for the culture of stem/progenitor cells

Hematopoietic Stem Cells

The definitive hematopoietic system is made up of all adult blood cell types

including megakaryocytes, erythrocytes, and cells of the myeloid and

lymphoid lineages. All of these cells are derived from multipotent

hematopoietic stem cells (HSCs) through a succession of precursors with

progressively limited potential. Hematopoietic stem cells are tissue-specific

stem cells that exhibit remarkable self-renewal capacity and are responsible

for the life-long maintenance of the hematopoietic system. HSCs are rare

cells that reside in adult bone marrow where hematopoiesis is continuously

taking place. They can also be found in cord blood, fetal liver, adult spleen, and peripheral blood. R&D Systems offers

several products for studying hematopoietic lineage cells including serum-free media, lineage depletion antibodies and kits,

and reagents for performing colony forming cell (CFC) assays.

Serum-Free Media for Hematopoietic Stem Cells

Culture media specifically optimized for use with hematopoietic stem cells.

A Summary of Hematopoietic Stem Cell Molecules

Supplements for Hematopoietic Stem Cell Expansion

A cytokine panel specifically designed for the support and expansion of hematopoietic stem cells.

Hematopoietic Lineage Depletion

Lineage depletion antibodies and kits that can be used to enrich for uncommitted cell populations.

Methylcellulose-based Reagents for Colony Forming Cell Assays

An in vitro quantitative assay used in the study of mouse and human hematopoietic stem cells.

Multi-Color Flow Cytometry Kits for Hematopoietic Stem Cells

Kits to simultaneously label cells using CD244-Fluorescein, CD150-APC, and CD48-PE

Serum-Free Media for Hematopoietic Stem Cells

StemXVivo™ Media for Cell Culture

Culture media specifically optimized for use with hematopoietic stem cells.

Researchers employing stem or progenitor cells in their studies currently use a wide variety of media products to support

their cultures. These media are generally developed or validated for cells other than stem cells, thus making them less

desirable than cell type-specific media. To address this need, R&D Systems offers StemXVivo media products specifically

designed for stem/progenitor cell culture. All the components included within these defined, basal, serum-free formulations

have been selected, optimized, and validated for hematopoietic stem cell culture.

Serum-Free Dendritic Cell Base Media (Catalog # CCM003)

Base Media optimized for the culture and differentiation of human dendritic cells.

Serum-Free T Cell Base Media (Catalog # CCM010)

Base Media optimized for the ex vivo culture of human T lymphocytes.

Dendritic Cell Culture and Maturation from CD14+ Monocytes. A. LPS-matured monocyte-derived dendritic cells

were obtained after a 9-day culture of CD14-enriched monocytes in StemXVivo Serum-Free Dendritic Cell Base Media

(Catalog # CCM003) supplemented with GM-CSF (Catalog # 215-GM) and IL-4 (Catalog # 204-IL). B. Dendritic cells

cultured for 7 days were stained with the mature dendritic cell marker CD83 before (gray line) and after a two day

treatment with LPS (black line). Isotype-matched control staining is also shown (filled histogram).

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Supplements for Hematopoietic Stem Cell Expansion

Mouse Hematopoietic Stem Cell Expansion Cytokine Panel (Catalog # SMPK9)

Hematopoietic stem cells (HSC) are well-characterized tissue-specific stem cells that exhibit remarkable self-renewal

capacity and are responsible for the life-long maintenance of the hematopoietic system. The combined use of multiple

cytokines (Flt-3 Ligand, SCF, and Tpo) permits the ex vivo expansion of HSC. The panel includes sufficient cytokines to

make 1000 mL of medium for cell expansion.

Kit Contents*

← Flt-3 Ligand

← SCF

← Tpo

*This kit requires media (not included).

Mouse Hematopoietic Stem Cell Expansion Cytokine PanelCatalog Number: SMPK9-CS

Catalog #: SMPK9ComponentCatalog #Amount Provided*Mouse Flt-3 Ligand427-FL100 μgMouse SCF455-MC100 μgMouse Tpo488-TO100 μg*Sufficient for 1 liter of media.

Mouse Cytokine Panel for Hematopoietic Stem Cell Expansion

← Hematopoietic stem cells (HSC) are well-characterized tissue-specific stem cells that exhibit remarkable self-

renewal capacity and are responsible for the life-long maintenance of the hematopoietic system. HSC are rare cells that

reside in adult bone marrow where hematopoiesis is continuously taking place. They can also be found in cord blood,

fetal liver, adult spleen and peripheral blood. Many HSC studies rely on ex vivo expansion of HSC in culture. The

combined use of multiple cytokines, Flt-3 ligand, SCF and Tpo, permits the ex vivo expansion of HSC. (800) 343-7475

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Hematopoietic Lineage Depletion

Lineage Depletion Antibodies & Kits

Lineage depletion antibodies and kits can be used to enrich for uncommitted cell populations.

Lineage Depletion Antibodies (Catalog # MLDP1 to MLDP8)

R&D Systems offers a range of lineage marker antibodies optimized to sufficiently bind 1 x 109 bone marrow derived cells.

These antibodies can conveniently be used in conjunction with magnetic particle separation systems (Catalog # MAG997)

or flow cytometric cell sorting to deplete lineage-committed cells for the enrichment of mouse uncommitted

mesenchymal or hematopoietic stem cells.

Analyte Type Clone #Catalog

#

Mouse B220/CD45R Monoclonal Rat IgG2B RA3-6B2 MLDP7

Mouse CD3 Monoclonal Rat IgG2B 17A2 MLDP1

Mouse CD4 Monoclonal Rat IgG2B GK1.5 MLDP2

Mouse CD5 Monoclonal Rat IgG2B 53-7.3 MLDP3

Mouse CD8 alpha Monoclonal Rat IgG2B 52-6.7 MLDP4

Mouse Integrin alpha M/CD Hb Monoclonal Rat IgG2B M1/70 MLDP5

Mouse Gr-1/Ly-6G Monoclonal Rat IgG2B RB6-8C5 MLDP6

Mouse TER-119 Monoclonal Rat IgG2B TER-119 MLDP8

MagCellect™ Mouse Hematopoietic Cell Lineage Depletion Kit** (Catalog # MAGM209)

Designed for the enrichment of hematopoietic non-lineage committed cells (lineage negative) from mouse bone marrow.

Lineage committed cells (lineage positive) targeted for depletion include T cells, B cells, NK cells, monocytes/macrophages,

granulocytes, and erythrocytes. The resulting cell population is highly enriched for SCF R/c-kit/CD117+ cells to a purity of 40-

70% (depending on mouse strain) with less than 5% residual lineage positive cells. This kit is for use with the MagCellect

Magnet; sold separately (Catalog # MAG997)

Kit Contents

← 10X Buffer

← Streptavidin Ferrofluid

← Blocking Reagent

← Anti-CD5

← Anti-TER-119

← Anti-CD11b

← Anti-CD45R/B220

← Anti-Ly-6G/Gr-1

Hematopoietic Lineage Depletion. Lineage marker

reactivity on BALB/c bone marrow (BM) cells processed with the

Enrichment of CD117+ Cells. CD117 (c-kit) staining of bone

marrow lineage negative cells isolated from BALB/c mice using

MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit

(Catalog # MAGM209). Histograms show reactivity of BM cells

labeled with the cocktail of biotinylated antibodies included in

the kit both before (green histogram) and after (open his-

togram) magnetic depletion. Lineage marker reactivity was

detected using Streptavidin-PE.

the MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit

(Catalog # MAGM209). Histograms reflect reactivity of all

viable cells with anti-CD117-PE (Catalog # FAB1356P; light

green) or a matched isotype control antibody (open histogram).

Inset shows CD117 staining of cells before depletion.

Mouse Hematopoietic Cell Lineage Depletion Protocol

**This product utilizes and/or contains technology licensed from Veridex, LLC, Raritan, New Jersey 08869, USA, which is covered by one or more claims of United States and International patents and/or pending patent applications.

Methylcellulose-based Reagents for Colony Forming Cell Assays

← Procedure for the Human Colony Forming Cell (CFC) Assay Using Methylcellulose-based Media

← Procedure for the Mouse Colony Forming Cell (CFC) Assay Using Methylcellulose-based Media

Methylcellulose Stock Solution (Catalog # HSC001)

Contains 3% methylcellulose in Iscove's MDM and can be used in both human and mouse hematopoietic stem cell research.

Human Methylcellulose Base Media (Catalog # HSC002)

Contains all of the basic components with the exception of the cytokines required to perform human CFC assays for

hematopoietic stem cell research.

Human Methylcellulose Serum-Free Base Media (Catalog # HSC002SF)

Serum-free media that contains all of the basic components with the exception of the cytokines required to perform human

CFC assays for hematopoietic stem cell research.

Mouse Methylcellulose Base Media (Catalog # HSC006)

Contains all of the basic components with the exception of the cytokines required to perform mouse CFC assays for

hematopoietic stem cell research.

Human Methylcellulose Complete Media (Catalog # HSC003

A specially formulated media supplemented with recombinant human (rh) GM-CSF, rhIL-3, rhSCF, and rhEpo optimized for

CFC assays of mouse clonogenic hematopoietic progenitors from human bone marrow, peripheral blood, cord blood,

leukopheresis products, and purified CD34+ cells.

Mouse Methylcellulose Complete Media (Catalog # HSC007)

A specially formulated media supplemented with recombinant mouse (rm) IL-3, rmIL-6, rmSCF, and recombinant human

Epo optimized for CFC assays of mouse clonogenic hematopoietic progenitors from mouse bone marrow, peripheral blood,

spleen, and fetal liver.

Human Methylcellulose Enriched Media (Catalog # HSC005)

A specially formulated media supplemented with recombinant human (rh) GM-CSF, rhG-CSF, rhIL-3, rhIL-6, rhSCF, and

rhEpo optimized for use with purified CD34+ cells in the CFC assay at the end of the long-term culture-initiating cell (LTC-IC)

assay.

Human Methylcellulose Serum-Free Enriched Media (Catalog # HSC005SF)

A specially formulated serum-free media supplemented with recombinant human (rh) GM-CSF, rhG-CSF, rhIL-3, rhIL-6,

rhSCF, and rhEpo optimized for CFC assays of human clonogenic hematopoietic progenitors from human bone marrow,

perihperal blood, cord blood, leukopheresis products, and purified CD34+ cells.

The products listed above are for the enumeration of:

← CFU-E (colony-forming unit-erythroid)

← BFU-E (burst-forming unit-erythroid)

← CFU-G (colony-forming unit-granulocyte)

← CFU-M (colony-forming unit-macrophage)

← CFU-GM (colony-forming unit-granulocyte/macrophage)

← CFU-GEMM (colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte)

Human Methylcellulose Complete Media without Epo (Catalog # HSC004)

A specially formulated media supplemented with recombinant human (rh) GM-CSF, rhIL-3, and rhSCF optimized for CFC

assays of human clonogenic hematopoietic progenitors from human bone marrow, peripheral blood, cord blood,

leukopheresis products, and purified CD34+ cells.

Human Methylcellulose Serum-Free Enriched Media without Epo (Catalog # HSC010SF)

A specially formulated serum-free media supplemented with recombinant human (rh) GM-CSF, rhG-CSF, rhIL-3, rhIL-6, and

rhSCF optimized for CFC assays of human clonogenic hematopoietic progenitors from human bone marrow, peripheral

blood, cord blood, leukopheresis products, and purified CD34+ cells.

Mouse Methylcellulose Complete Media without Epo (Catalog # HSC008)

A specially formulated media supplemented with recombinant mouse (rm) IL-3, rmIL-6, and rmSCF optimized for CFC

assays of mouse clonogenic hematopoietic progenitors from mouse bone marrow, peripheral blood, spleen, and fetal liver.

The products listed above are for the enumeration of:

← CFU-GM

← CFU-G

← CFU-M

Examples of colony types

CFU-E (Colony forming unit-erythroid):

Clonogenic progenitors that produce only one or

two clusters with each cluster containing from 8 to

approximately 100 hemoglobinized erythroblasts.

It represents the more mature erythroid

progenitors that have less proliferative capacity.

CFU-G (Colony forming unit-

granulocyte): Clonogenic progenitors of

granulocytes that give rise to a

homogeneous population of eosinophils,

basophils or neutrophils.

CFU-GM (Colony forming unit-granulocyte,

macrophage): Progenitors that give rise to

colonies containing a heterogeneous population

of macrophages and granulocytes. The mor-

phology is similar to the CFU-M and CFU-G

descriptions.

BFU-E (Burst forming unit-erythroid): The size

of the colony can be described as small (3 to 8

clusters), intermediate (9 to 16 clusters), or large

(more than 16 clusters) according to the number

of clusters present. These are primitive erythroid

progenitors that have high proliferative capacity.

CFU-M (Colony forming unit-

macrophage): Clonogenic progenitors of

macrophages that give rise to a ho-

mogenous population of macrophages.

CFU-GEMM (Colony forming unit-

granulocyte, erythrocyte,macrophage,

megakaryocyte): Multi-lineage progenitors

that give rise to erythroid, granulocyte,

macrophage and megakaryocyte lineages, as

the name indicates.

Catalog Numbers link to Product Data sheets and require Adobe Acrobat Reader

DESCRIPTIONANTIBODY

APPLICATION (KEY)CATALOG #/DATASHEET SIZE PRICE

Mouse Hematopoietic Progenitor Cell 3-Color Flow Kit

Contains conjugated antibodies to CD244-Fluorescein (Goat IgG), CD150-APC (Clone

459911), CD48-PE (Clone 331504)

StemXVivo™ Culture Matrix

StemXVivo Culture Matrix (Catalog # CCM013) is a defined proprietary mixture of recombinant human adhesion molecules

for the culture of stem/progenitor cells. It can be used as a substitute for EHS basement membrane extract (such as

Matrigel™) or as a feeder layer in the maintenance and/or differentiation of stem/progenitor cells. The culture matrix is

tested for its ability to support attachment and growth of multiple stem/progenitor cell populations.

Oct-3/4 and Nestin were detected in Human TE03 Cells. Human TE03 Embryonic Stem Cells were grown either in StemXVivo Culture Matrix (Catalog #

CCM013) or EHS basement membrane extract. Oct-3/4 was detected using Human/Mouse Oct-3/4 Monoclonal Antibody (MAB1759; red) and Nestin was detected

using Nestin Polyclonal Antibody (green). Cells were stained and the nuclei were counterstained with DAPI (blue). Data courtesy of Dr. Ronald McKay, NINDS.

StemXVivo is a trademark of R&D Systems, Inc.Matrigel is a trademark of Becton Dickinson and Company.

Extracellular Matrix Pre-coated Plates

R&D Systems offers a range of aseptically prepared microplates pre-coated with extracellular matrix (ECM) proteins for

culturing adherent cell lines and quantifying cell adhesion.

Many proteins of the ECM interact with cells via cell surface integrin family receptors. The resulting focal contacts are

important for the maintenance of tissue architecture and for supporting a variety of cellular processes. ECM protein/integrin

binding may initiate a complex network of signal transduction cascades that, depending on the context, plays an important

role in cell spreading, migration, proliferation, and differentiation during embryogenesis, wound healing, and tumor

development.

Extracellular Matrix Pre-coated Plates

Kits are available containing 96-well microplates pre-coated with Fibronectin (Catalog # CWP001, CWP002) or Vitronectin

(Catalog # CWP003, CWP004). These aseptic plates may be used to promote cell attachment during culture of adherent cell

lines.

Cell Adhesion Assays

Microplates pre-coated with ECM proteins can also be used as a tool to quantify cell adhesion. Cell suspensions are simply

applied to pre-coated microplates and centrifuged to promote interaction with the plate surface. Non-adherent cells are

gently removed during PBS washes. The percentage of adherent cells is determined by measuring Calcein AM fluorescence

before and after the wash steps.

Product Features

← Label: Calcein AM.*

← Testing Format: Fluorescence microplate reader at 485nm and 520nm.

← Sample Type: Cell suspensions.

← Size: 5 x 96-well microplates.

*This assay requires additional reagents; Calcein AM (Catalog # 4892-010-K) and PBS with calcium and magnesium.

Figure 1. R&D Systems Extracellular Matrix (ECM) Protein Coated Plate assay template (left). As

indicated by text in wells, rows A and H are left uncoated to serve as controls, and rows B through G are

coated with ECM protein. The entire plate is then blocked with 1% Bovine Serum Albumin Fraction V

(BSA). In this assay, cells were either unstimulated (burgundy) or stimulated (orange) with 10 ng/mL PMA

for 30 minutes and applied to R&D Systems Human Fibronectin Coated Plates (Catalog # CWP001) in the

pattern shown. Cells were labeled with calcein-AM and adhesion to the plates was measured as a

percentage of total cells applied (right). All bars depict average and standard error values obtained from

three plates.

Extracellular Matrix Molecules - Coat your own plates with R&D Systems recombinant ECM proteins.

Extracellular Matrix-Associated Molecules

Basement Membrane Extracts - An alternative approach to promote cell adhesion.

Basement Membrane Extracts

← Complete Listing of Basement Membrane Extracts

Basement Membrane Extracts promote and maintain differentiated phenotypes of cells in culture.

Basement membranes are continuous sheets of specialized extracellular matrix that form an interface between endothelial,

epithelial, muscle, or neuronal cells and their adjacent stroma. Cultrex® Basement Membrane Extract (BME) is a soluble

basement membrane extract of the Engelbreth-Holm-Swarm (EHS) tumor. All Cultrex Basement Membrane Extracts

undergo vigorous sterility testing with no bacteria or fungi detected after incubation at 37° C for 14 days. They are negative

for mycoplasma by PCR and the Endotoxin level is XX 20 EU/mL by LAL. These specialized solutions gel within 30 mins at

37° C to form a reconstituted basement membrane on any desired growth surface. Use BME to promote and maintain a

differentiated phenotype of cell cultures such as epithelial cells, endothelial cells, glandular cells, neurons, and smooth

muscle cells.

Product features

← Rapid - gels within 30 minutes at 37º C.

← Reliable -maintains gelled form in culture medium for 14 days.

← Sterile - screened for bacteria, fungi, and mycoplasma.

← Specific - available with reduced growth factor content, with or without Phenol Red, and additional sterility testing.

Cultrex Basement Membrane Extract

BME consists of laminin I, type IV collagen, entactin, and heparan sulfate proteoglycan.

Cultrex Basement Membrane Extract - without Phenol Red

Since Phenol Red has been shown to act as a differentiating factor and may affect some colorimetric or fluorescence-based

assays, extracts are available without Phenol Red.

Cultrex Basement Membrane Extract – Reduced Growth Factor

For studies focused on cell growth, BME is subjected to a special process to reduce growth factor content.

Growth Factor BME (standard) BME (reduced growth factor)

bovine FGF 0-0.1 pg/mL 0-0.1 pg/mL

human EGF 0.5-13 ng/mL < 0.5 ng/mL

human IGF-I 15.6 ng/mL 5.0 ng/mL

human PDGF 12 pg/mL < 5.0 pg/mL

human NGF < 0.2 ng/mL < 0.2 ng/mL

human TGF-beta 2.3 ng/mL 1.7 ng/mL

Cultrex Basement Membrane Extract – PathClear®

PathClear products undergo additional testing and are confirmed negative by PCR for 31 infectious organisms and viruses,

including the LDEV virus.

Cultrex 3-D Culture Matrix™

Specifically produced for and qualified in 3-D culture studies.

Cultrex Basement Membrane Extract consists of laminin I, type IV collagen, entactin, and heparan sulfate proteoglycan.

Basement Membrane Extracts specific for individual basement membrane proteins of interest are also available.

Basement Membrane-Specific Extracts

← Mouse Laminin I (Catalog # 3400-010-01)

← Bovine Collagen I (Catalog # 3442-050-01)

← Rat Collagen I (Catalog # 3440-100-01)

← Mouse Collagen IV (Catalog # 3410-010-01)

← Cultrex Cell Staining Kit (Catalog # 3437-100-K)

The Cell Staining solution contains a mixture of Azur A and Methylene Blue, specially formulated to provide optimized

staining of cells and structures grown on Basement Matrix Extract.

BME Cell Invasion Assays

Fluorescence-based 96-well assays for investigating chemotaxis, cell migration, and cell invasion.

Cultrex and PathClear are registered trademarks of Trevigen, Inc.3-D Culture Matrix is a trademark of Trevigen, Inc.

Cell Invasion Assays

← Complete Listing of Cell Invasion Assays

Fluorescence-based 96-well assays for investigating chemotaxis, cell migration, and cell invasion.

The Cultrex® Cell Invasion Assay is designed to accelerate the screening process for compounds that influence cell

migration through extracellular matrices. Invasive migration is a fundamental function underlying cellular processes such as

angiogenesis, embryonic development, immune response, metastasis, and invasion of cancer cells. This assay offers a

flexible, standardized, high-throughput format for quantitating the degree to which invasive cells penetrate a barrier

consisting of basement membrane components in response to chemoattractants and/or inhibiting compounds.

Cultrex BME Cell Invasion Assay (Catalog # 3455-096-K)

This 96-well assay includes Cultrex BME, which consists of laminin I, type IV collagen, entactin, and heparan sulfate

proteoglycan. Cell suspensions are applied to BME-coated cell invasion chambers (included in kit). The invasive capacity of

a given cell sample is determined by measuring Calcein AM fluorescence.

Kit Contents

← Cell Invasion Chamber

← BME Solution*

← Coating Buffer

← Cell Wash Buffer

← Cell Dissociation Solution

← Calcein AM

*Kits are also available to test cell invasion using barriers that are composed specifically of Laminin I (Catalog # 3456-096-K), Collagen I (Catalog # 3457-096-K), and Collagen IV (Catalog # 3458-096-K).

Since different cell lines and different treatments can result in a wide range of invasive potentials, the permissiveness of

the matrix may be optimized for each experiment by adjusting the coating concentration

Illustration of a Cell Invasion Assay. The invasion chamber consists of

two chambers separated by a filter coated with BME or different ECM

components. The cell suspension is placed in the top chamber, and

incubated in the presence of test media containing specific

chemoattractants in the bottom chamber. Cells migrate from the top

chamber through the coated filter pores to the bottom of the filter. Cell

dissociation/Calcein AM solution is placed in the bottom chamber to

dissociate the migrating cells from the filter and add a fluorescent label.

Fluorescence in the bottom chamber is proportional to the number of

migrating cells.

 

Quantification of Cell Invasion. Cultrex Cell Invasion Assay

Kits (Catalog # 3455-096-K, 3456-096-K, 3457-096-K, 3458-

096-K) were used to quantify the ability of 10% FBS to

stimulate the migration of fibroblastic cell lines on different

extracellular matrix components. Data from four experiments

was quantified for both non-invasive (NIH-3T3) and invasive

(HT-1080) cell types.

 

Cultrex is a registered trademark of Trevigen, Inc.

Mycoplasma Detection

← Complete Listing of Mycoplasma Detection Kits

A highly sensitive colorimetric microplate-based assay designed to detect the eight mycoplasma species known to cause

95% of eukaryotic cell culture contamination.

R&D Systems MycoProbe™ Mycoplasma Detection Kit (Catalog # CUL001B) permits high-throughput, routine screening of

cell cultures for mycoplasma contamination. Mycoplasma contamination is one of the most common and serious problems

when culturing eukaryotic cells. Mycoplasma contamination has the potential to alter the phenotypic characteristics of the

cells and can negatively impact results. Since mycoplasma is typically not visible and does not respond to antibiotics, an

alternative, sensitive, and reliable detection method is required.

MycoProbe Mycoplasma Detection Kit (Catalog # CUL001B)

Assay Features

← High-throughput screening (microplate-based) assay

← No false positives from amplicon contamination

← Highly sensitive (comparable to PCR)

← Culturing in antibiotic-free media is not required

← Pellet or supernate sample may be used from fresh or frozen cells

← Rapid results in 4.5 hours

← Reagents sufficient for 96 tests

Kit Contents

Each MycoProbe Mycoplasma Detection Kit contains all of the necessary components for a ready-to-run assay and requires

no additional development. Each kit contains sufficient reagents for 96 tests. We recommend running samples in duplicate

with positive and negative controls.

← Cell Lysis Buffer

← Hybridization Plate

← Streptavidin Capture Plate

← Diluents

← Substrate

← Conjugate

← Pan-Specific Oligonucleotide Probes

← Synthetic DNA Oligonucleotide Positive Control

Mycoplasma Bacteria Detected:

← M. hyorhinis

← M. arginini

← M. fermentans

← M. orale

← M. pirum

← M. hominis

← M. salivarium

← A. laidlawii

What are Stem Cells?

Stem cells are a class of undifferentiated cells that are able to differentiate into specialized cell types. Commonly, stem cells come from two main sources:

1. Embryos formed during the blastocyst phase of embryological development (embryonic stem cells) and 2. Adult tissue (adult stem cells).

Both types are generally characterized by their potency, or potential to differentiate into different cell types (such as skin, muscle, bone, etc.).

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.

Embryonic stem cells

Embryonic stem cells are derived from a four- or five-day-old human embryo that is in the blastocyst phase of development. The embryos are usually extras that have been created in IVF (in vitro fertilization) clinics where several eggs are fertilized in a test tube, but only one is implanted into a woman.

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).

9-week Human Embryo from Ectopic Pregnancy [by Ed Uthman, MD]creative commons license

In a normal pregnancy, the blastocyst stage continues until implantation of the embryo in the uterus, at which point the embryo is referred to as a fetus. This usually occurs by the end of the 10th week of gestation after all major organs of the body have been created.

However, when extracting embryonic stem cells, the blastocyst stage signals when to isolate stem cells by placing the "inner cell mass" of the blastocyst into a culture dish containing a nutrient-rich broth. Lacking the necessary stimulation to differentiate, they begin to divide and replicate while maintaining their ability to become any cell type in the human body. Eventually, these undifferentiated cells can be stimulated to create specialized cells.

Stem cell cultures

Human embryonic stem cell colony[Wikipedia]

Stem cells are either extracted from adult tissue or from a dividing zygote in a culture dish. Once extracted, scientists place the cells 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, but progress is being made for both cell types.

Stem cell lines

Once stem cells have been allowed to divide and propagate in a controlled culture, the collection of healthy, dividing, and undifferentiated cells is called a stem cell line. These stem cell lines are subsequently managed and shared among researchers. Once under control, the stem cells can be stimulated to specialize as directed by a researcher - a process known as directed differentiation. Embryonic stem cells are able to differentiate into more cell types than adult stem cells.

Potency

Stem cells are categorized by their potential to differentiate into other types of cells. Embryonic stem cells are the most potent since they must become every type of cell in the body. The full classification includes:

Totipotent - the ability to differentiate into all possible cell types. Examples are the zygote formed at egg fertilization and the first few cells that result from the division of the zygote.

Pluripotent - the ability to differentiate into almost all cell types. Examples include embryonic stem cells and cells that are derived from the mesoderm, endoderm, and ectoderm germ layers that are formed in the beginning stages of embryonic stem cell differentiation.

Multipotent - the ability to differentiate into a closely related family of cells. Examples include hematopoietic (adult) stem cells that can become red and white blood cells or platelets.

Oligopotent - the ability to differentiate into a few cells. Examples include (adult) lymphoid or myeloid stem cells. Unipotent - the ability to only produce cells of their own type, but have the property of self-renewal required to

be labeled a stem cell. Examples include (adult) muscle stem cells.

Embryonic stem cells are considered pluripotent instead of totipotent because they do not have the ability to become part of the extra-embryonic membranes or the placenta.

What are stem cells - Video

A video on how stem cells work and develop.

Identification of stem cells

Although there is not complete agreement among scientists of how to identify stem cells, most tests are based on making sure that stem cells are undifferentiated and capable of self-renewal. Tests are often conducted in the laboratory to check for these properties.

One way to identify stem cells in a lab, and the standard procedure for testing bone marrow or hematopoietic stem cell (HSC), is by transplanting one cell to save an individual without HSCs. If the stem cell produces new blood and immune cells, it demonstrates its potency.

Clonogenic assays (a laboratory procedure) can also be employed in vitro to test whether single cells can differentiate and self-renew. Researchers may also inspect cells under a microscope to see if they are healthy and undifferentiated or they may examine chromosomes.

To test whether human embryonic stem cells are pluripotent, scientists allow the cells to differentiate spontaneously in cell culture, manipulate the cells so they will differentiate to form specific cell types, or inject the cells into an immunosuppressed mouse to test for the formation of a teratoma (a benign tumor containing a mixture of differentiated cells).

Research with stem cells

Scientists and researchers are interested in stem cells for several reasons. Although stem cells do not serve any one function, many have the capacity to serve any function after they are instructed to specialize. Every cell in the body, for example, is derived from first few stem cells formed in the early stages of embryological development. Therefore, stem

cells extracted from embryos can be induced to become any desired cell type. This property makes stem cells powerful enough to regenerate damaged tissue under the right conditions.

Organ and tissue regeneration

Tissue regeneration is probably the most important possible application of stem cell research. Currently, organs must be donated and transplanted, but the demand for organs far exceeds supply. Stem cells could potentially be used to grow a particular type of tissue or organ if directed to differentiate in a certain way. Stem cells that lie just beneath the skin, for example, have been used to engineer new skin tissue that can be grafted on to burn victims.

Brain disease treatment

Additionally, replacement cells and tissues may be used to treat brain disease such as Parkinson's and Alzheimer's by replenishing damaged tissue, bringing back the specialized brain cells that keep unneeded muscles from moving. Embryonic stem cells have recently been directed to differentiate into these types of cells, and so treatments are promising.

Cell deficiency therapy

Healthy heart cells developed in a laboratory may one day be transplanted into patients with heart disease, repopulating the heart with healthy tissue. Similarly, people with type I diabetes may receive pancreatic cells to replace the insulin-producing cells that have been lost or destroyed by the patient's own immune system. The only current therapy is a pancreatic transplant, and it is unlikely to occur due to a small supply of pancreases available for transplant.

Blood disease treatments

Adult hematopoietic stem cells found in blood and bone marrow have been used for years to treat diseases such as leukemia, sickle cell anemia, and other immunodeficiencies. These cells are capable of producing all blood cell types, such as red blood cells that carry oxygen to white blood cells that fight disease. Difficulties arise in the extraction of these cells through the use of invasive bone marrow transplants. However hematopoietic stem cells have also been found in the umbilical cord and placenta. This has led some scientists to call for an umbilical cord blood bank to make these powerful cells more easily obtainable and to decrease the chances of a body's rejecting therapy.

General scientific discovery

Stem cell research is also useful for learning about human development. Undifferentiated stem cells eventually differentiate partly because a particular gene is turned on or off. Stem cell researchers may help to clarify the role that genes play in determining what genetic traits or mutations we receive. Cancer and other birth defects are also affected by abnormal cell division and differentiation. New therapies for diseases may be developed if we better understand how these agents attack the human body.

Another reason why stem cell research is being pursued is to develop new drugs. Scientists could measure a drug's effect on healthy, normal tissue by testing the drug on tissue grown from stem cells rather than testing the drug on human volunteers.

Stem cell controversy

The debates surrounding stem cell research primarily are driven by methods concerning embryonic stem cell research. It was only in 1998 that researchers from the University of Wisconsin-Madison extracted the first human embryonic stem cells that were able to be kept alive in the laboratory. The main critique of this research is that it required the destruction of a human blastocyst. That is, a fertilized egg was not given the chance to develop into a fully-developed human.

When does life begin?

The core of this debate - similar to debates about abortion, for example - centers on the question, "When does life begin?" Many assert that life begins at conception, when the egg is fertilized. It is often argued that the embryo deserves the same status as any other full grown human. Therefore, destroying it (removing the blastocyst to extract stem cells) is akin to murder. Others, in contrast, have identified different points in gestational development that mark the beginning of life - after the development of certain organs or after a certain time period.

Chimeras

People also take issue with the creation of chimeras. A chimera is an organism that has both human and animal cells or tissues. Often in stem cell research, human cells are inserted into animals (like mice or rats) and allowed to develop. This creates the opportunity for researchers to see what happens when stem cells are implanted. Many people, however, object to the creation of an organism that is "part human".

Legal issues

The stem cell debate has risen to the highest level of courts in several countries. Production of embryonic stem cell lines is illegal in Austria, Denmark, France, Germany, and Ireland, but permitted in Finland, Greece, the Netherlands, Sweden, and the UK. In the United States, it is not illegal to work with or create embryonic stem cell lines. However, the debate in the US is about funding, and it is in fact illegal for federal funds to be used to research stem cell lines that were created after August 2001.

Stem cell research news

Medical News Today is a leading resource for the latest headlines on stem cell research. So, check out our stem cell research news section. You can also sign up to daily stem cell news alerts or our weekly digest newsletters to ensure that you stay up-to-date with the latest news.

Stem Cell & Cell Culture Products

R&D Systems offers a range of specialized tools for culturing and maintaining a wide variety of cell types under both serum

and serum-free conditions.

Stem Cell Products

R&D Systems offers a wide range of products for the study of embryonic, neural, mesenchymal, and hematopoietic stem

cells. We provide primary cells, culture media, and kits for expansion and differentiation. In addition, we offer a variety of

products for assessing the differentiation status of cell lines based on the expression of lineage-specific protein markers.

Embryonic & Induced Pluripotent Stem Cells

iMEFs, conditioned media, and marker panels to maintain, expand, and monitor differentiation of ESCs and iPS.

Neural Stem Cells

Cortical stem cells, along with media, expansion kits, and functional identification kits for directing and characterizing cells

along the neuronal lineage.

Mesenchymal Stem Cells

Media, supplements, and functional identification kits for promoting and monitoring MSC progression to osteogenic,

adipogenic, or chondrogenic lineages.

Hematopoietic Stem Cells

Media, supplements, and enrichment reagents to study HSCs, as well as colony forming cell assay reagents.

Stem Cell Antibody Arrays

Simultaneous detection of 15 pluripotent stem cell markers without the need for specialized equipment.

StemXVivo™ Culture Matrix

A defined proprietary mixture of recombinant human adhesion molecules for the culture of stem/progenitor cells

Cell Culture Products

Extracellular Matrix Pre-coated Plates

Pre-coated microplates for culturing adherent cell lines and quantifying cell adhesion.

Extracellular Matrix Molecules

Recombinant Extracellular Matrix (ECM) proteins for promoting cell adhesion.

Basement Membrane Extracts

Form continuous sheets of specialized extracellular matrix that provide an interface between endothelial, epithelial, muscle,

or neuronal cells and their adjacent stroma.

Cell Invasion Assays

Fluorescence-based assays for investigating chemotaxis, cell migration, and cell invasion.

Mycoplasma Detection

A highly sensitive, high-throughput microplate-based assay designed to detect eukaryotic cell culture contamination.

Cell Culture Media Supplements & Reagents

Additional supplies for cell culture.

Embryonic & Induced Pluripotent Stem Cells

Embryonic stem (ES) cells, which are derived from the inner cell mass of pre-

implantation embryos, have been recognized as the most pluripotent stem cell

population. These cells are capable of unlimited, undifferentiated proliferation in

vitro and still maintain the capacity for differentiation into a wide variety of

somatic tissues. Induced Pluripotent Stem Cells (iPS) are generated from somatic

cells following the expression of specific genes. Both types of stem cells have

widespread clinical potential in the treatments of heart disease, diabetes, spinal

cord injury, and a variety of neurodegenerative disorders. R&D Systems offers a range of products designed to maintain

and expand stem cells in culture as well as monitor their differentiation status.

A Summary of Embryonic Stem Cell Molecules

Feeder Cells for Embryonic & Induced Pluripotent Stem Cell Culture

Mouse embryonic fibroblasts are used to maintain and expand pluripotent human embryonic stem cells (hES) in an

undifferentiated state or to induce differentiation along somatic or germ cell lineages.

Conditioned Media for Embryonic & Induced Pluripotent Stem Cell Culture

Culture media specifically optimized for use with human or rodent embryonic stem cells.

Stem Cell Antibody Arrays

Simultaneous detection of 15 pluripotent stem cell markers without the need for specialized equipment.

Multi-Color Flow Cytometry Kits for Embryonic Stem Cells

Kits to simultaneously label cells using SSEA-1-PerCP, SSEA-4-Fluorescein, Oct-3/4-APC, and SOX2-PE.

StemXVivo™ Culture Matrix

A defined proprietary mixture of recombinant human adhesion molecules for the culture of stem/progenitor cells.

Marker Panels for Embryonic Stem Cells

R&D Systems offers a range of products to assess differentiation status and identify specific cell types of interest.

← Antibody Panels for Embryonic Stem Cells

← Primer Pair Panels for Embryonic Stem Cells

Irradiated Mouse Embryonic Fibroblasts (iMEF; Catalog # PSC001)

Mouse embryonic fibroblasts are used to maintain and expand pluripotent human embryonic stem cells (hES) in an

undifferentiated state or to induce differentiation along somatic or germ cell lineages. R&D Systems iMEFs (Catalog #

PSC001) are mitotically inactivated by irradiation rather than by use of pharmacological inhibitors, thus eliminating possible

exposure of your ES cells to toxic agents.

Each iMEF lot is confirmed negative for mycoplasma contamination using the MycoProbe® mycoplasma detection kit

(Catalog # CUL001B) and negative for microbial contamination. Furthermore, each lot is tested for its ability to support

undifferentiated growth of the BG01V human embryonic stem cells as assessed by Oct-3/4 and SSEA-4 expression.

Features

← Consistent

← Ready-to-use

← Mitotically inactivated by irradiation

← Maintains pluripotency of human ES cells

← Negative for mycoplasma and microbial contamination

A B

C D

Human Embryonic Stem Cells (Cell Line BG01V) Cultured on iMEFs were Assessed Using Markers of

Pluripotency. BG01V colonies (highlighted by arrows) growing in culture on iMEFs (A) were analyzed for markers of

pluripotency after 3 passages. Cells were evaluated for SSEA-4 expression by flow cytometry (B) using PE-conjugated

anti-human SSEA-4 (Catalog # FAB1435P; filled histogram) or isotype control antibody (open histogram). BG01V

colonies were incubated with anti-human Oct-3/4 (C, Catalog # AF1759) or anti-human Nanog (D), Catalog # AF1997)

followed by staining with NorthernLights™ 557-conjugated anti-goat secondary antibody (Catalog # NL001).

BG01V Cells are licensed from BresaGen, Inc.

Conditioned Media for Embryonic & Induced Pluripotent Stem Cells

Media for Cell Culture

Culture media specifically optimized for use with human and rodent stem and progenitor cells.

Researchers employing stem or progenitor cells in their studies currently use a wide variety of media products to support

their cultures. These media are generally developed or validated for cells other than stem cells, thus making them less

desirable than cell type-specific media. To address this need, R&D Systems offers a media product specifically designed for

stem/progenitor cell culture. All the components included within this defined, basal, serum-free formulation have been

selected, optimized, and validated for embryonic stem cell culture.

Mouse Embryonic Fibroblast Conditioned Media (Catalog # AR005)

Serum-free media (80% Knockout™ D-MEM, 20% Knockout serum replacement, 1% MEM non-essential amino acids, 2 mM

GlutaMAX™, 0.1 mM beta-mercaptoethanol, 4 ng/mL basic fibroblast growth factor (FGF basic)) was conditioned by gamma-

irradiated CF-1 fibroblasts at 37° C for 24 hours.

A

B SSEA-4 Expression in Embryonic Stem Cells. Human embryonic stem

cells were cultured with recombinant human FGF basic (Catalog # 233-FB)

in the presence (A) or absence (B) of Mouse Embryonic Fibroblast (MEF)-

conditioned media (Catalog # AR005). SSEA-4 and Oct-3/4 were detected

using anti-human SSEA-4 monoclonal antibody (red, Catalog # MAB1435)

and anti-human Oct-3/4 polyclonal antibody (green, Catalog # AF1759).

Cells were counterstained with DAPI (blue). Image courtesy of Dr. Frank

Soldner of the National Institutes of Health.

Knockout, GlutaMAX, and KSR are trademarks of Life Technologies, Carlsbad, CA.

GlobalStem ES-DMEM is a trademark of GlobalStem, Rockville, MD.

Human Pluripotent Stem Cell Antibody Array

← Human Pluripotent Stem Cell Antibody Array Ordering Information

Introduction

The Human Pluripotent Stem Cell Antibody Array is a rapid, sensitive, and economical tool to simultaneously detect the

relative expression of 15 pluripotent stem cell markers in a single sample. No specialized equipment is necessary.

← Product Insert (PDF)

← Troubleshooting Guide

← Assays for analytes represented in the Human

Pluripotent Stem Cell Array Kit

← Antibodies for analytes   related to   the Human

Pluripotent Stem Cell Array Kit

Simultaneously detect the relative levels of these pluripotent stem cell markers in a single sample.

alpha-Fetoprotein GATA-4 TP63/TP73L

Oct-3/4 HNF-3 beta/FoxA2 Goosecoid

Nanog PDX-1/IPF1 Snail

SOX2 SOX17 VEGF R2/KDR

E-Cadherin Otx2 HCG

General Assay Principle

Carefully selected capture antibodies have been spotted in

duplicate on nitrocellulose membranes. Cellular extracts are

diluted and incubated with the Human Pluripotent Stem Cell

Array overnight. The array is washed to remove unbound

proteins, followed by incubation with a cocktail of biotinylated

detection antibodies. Streptavidin-HRP and chemiluminescent

detection reagents* are applied, and a signal is produced at

each capture spot corresponding to the amount of protein

bound.

Kit Contents

← 8 Array Membranes

← 8-well Multi-dish

← Array Buffers

The Human Pluripotent Stem Cell Array detects multiple stem

cell markers in differentiated BG01V cell extracts. Arrays were

incubated with 200 µg of each cell extract shown above. Array images

shown were collected from 3 minute exposures to X-ray film. Labels

on the films correspond to the same proteins quantified on the bar

chart.

← Lysis Buffer

← Wash Buffer

← Antibody Detection Cocktail

← Streptavidin-HRP

← Transparency Overlay Template

← Detailed Protocol

*Chemiluminescence detection substrate not included. For a complete list of the kit contents and necessary materials, please see the Materials Provided/Materials Required sections of the product insert (PDF).

Human/Mouse Embryonic Stem Cell Multi-Color Flow Cytometry KitCatalog Number: FMC001Size: 25 TestsProduct DescriptionThis kit contains four conjugated antibodies (and corresponding isotype controls) that can be used for single-step staining of human/mouse embryonic stem cells (h/mESCs) (1 - 7):• SOX2-PE (Clone 245610; mouse IgG2A)• Oct-3/4-APC (Clone 240408; rat IgG2B)• SSEA-1-PerCP (Clone MC-480; mouse IgM)• SSEA-4-CFS (Clone MC-813-70; mouse IgG3)The kit also contains Fixation/Permeabilization Buffer (30 mL), which contains 1% formaldehyde, saponin, and < 0.05% sodium azide as well as Permeabilization/Wash Buffer (60 mL), which contains saponin and 0.05% sodium azide.Intended UseThis product is designed for the flow cytometric analysis of h/mESCs using four fluorochrome-conjugated antibodies.StorageStore at 2 - 8° C in the dark. Use within 6 months of receipt.Precautions• Formaldehyde is a suspected carcinogen. Avoid contact with skin, eyes, and mucous membranes, and avoid inhaling fumes. In case of contact, wash immediately with water and seek medical advice.

• Sodium azide may react with lead and copper plumbing to form explosive metallic azides. Flush with large volumes of water during disposal.Intracellular Staining Protocol with Simultaneous Fixation/Permeabilization1. Harvest cells of interest and wash twice in PBS or Hanks’ Balanced Salt Solution (HBSS).2. Approximately 5 x 105 washed cells should be resuspended in 0.5 mL of Fixation/Permeabilization Buffer and incubated at 2 - 8° C for 30 minutes. Cells should be vortexed intermittently in order to maintain a single cell suspension.3. The cells are centrifuged and the pellet is resuspended in 100 - 200 μL of the Permeabilization/Wash Buffer.4. Add 10 μL of each antibody or each corresponding isotype control antibody to the cells.5. Incubate the mixture for 30 - 45 minutes at 2 - 8° C in the dark.6. Following the incubation, remove any excess antibody by washing the cells in 2 mL of Permeabilization/Wash Buffer. The final cell pellet is resuspended in 200 - 400 μL of PBS for flow cytometric analysis. Notes: Because saponin-mediated cell permeabilization is a reversible process, it is important to keep the cells in the presence of saponin during intracellular staining. Using multiple fluorochromes requires proper flow cytometric compensation to remove the spillover fluorescence from a particular probe to a certain channel (8).

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. R&D Systems, Inc.1-800-343-7475726058.2 7/09

Typical DataFigure 1: Human BG01V embryonic stem cells were stained using the Human/Mouse Embryonic Stem Cell Multi-Color Flow Cytometry Kit (Catalog # FMC001). Cells were analyzed simultaneously for their expressions of SSEA-1, SSEA-4, Oct-3/4 and SOX2.Figure 2: Mouse D3 embryonic stem cells were stained using the Human/Mouse Embryonic Stem Cell Multi-Color Flow Cytometry Kit (Catalog # FMC001). Cells were analyzed simultaneously for their expressions of SSEA-1, SSEA-4, Oct-3/4 and SOX2.References1. Evans, M.J. and M.H. Kaufman (1981) Nature 292:154.2. Martin, G. (1981) Proc. Natl. Acad. Sci. USA 78:7634.3. Thomson, J.A. et al. (1998) Science 282(5391):1145.4. Rosner, M.H. et al. (1990) Nature 345:686.5. Niwa, H. et al. (2000) Nat. Genet. 24:372.6. Chambers, I. et al. (2003) Cell 113:643.7. Mitsui, K. et al. (2003) Cell 113:631.8. Bagwell, B. and E.G. Adams (1993) Ann. N.Y. Acad. Sci. 677:167.BG01V cells are licensed from Novocell, Inc.

StemXVivo™ Culture Matrix

StemXVivo Culture Matrix (Catalog # CCM013) is a defined proprietary mixture of recombinant human adhesion molecules

for the culture of stem/progenitor cells. It can be used as a substitute for EHS basement membrane extract (such as

Matrigel™) or as a feeder layer in the maintenance and/or differentiation of stem/progenitor cells. The culture matrix is

tested for its ability to support attachment and growth of multiple stem/progenitor cell populations.

Oct-3/4 and Nestin were detected in Human TE03 Cells. Human TE03 Embryonic Stem Cells were grown either in StemXVivo Culture Matrix (Catalog #

CCM013) or EHS basement membrane extract. Oct-3/4 was detected using Human/Mouse Oct-3/4 Monoclonal Antibody (MAB1759; red) and Nestin was detected

using Nestin Polyclonal Antibody (green). Cells were stained and the nuclei were counterstained with DAPI (blue). Data courtesy of Dr. Ronald McKay, NINDS.

StemXVivo is a trademark of R&D Systems, Inc.Matrigel is a trademark of Becton Dickinson and Company.

Antibody Panel for Embryonic Stem Cells

Embryonic Stem Cell Antibody Panels

These antibody panels are designed for analyzing the differentiation status of human embryonic stem (ES) cells by

evaluating ES cell marker expression. Each panel contains a group of antibodies that are recognized as markers for

undifferentiated human ES cells. Panels can be applied to both flow cytometry- and immunohistochemistry-based

applications.

While the undifferentiated/pluripotent state of ES cells can be best defined functionally, a number of molecular markers

have been used to characterize it. Pluripotent ES cells can be characterized by high level expression of the transcription

factors Oct-3/4 (also termed Oct-3 or Oct-4) and Nanog. A critical amount of Oct-3/4 and Nanog expression is required to

sustain stem-cell pluripotency. When ES cells are induced to differentiate, Oct-3/4 and Nanog are down-regulated.

The undifferentiated state of ES cells is often characterized by the expression of the cell surface antigens, SSEA-1 and

SSEA-4. Undifferentiated primate ES cells, human ES cells, and human Embryonic Carcinoma (EC) cells express SSEA-4, but

not SSEA-1. In contrast, undifferentiated mouse ES cells do express SSEA-1 but do not express SSEA-4. Undifferentiated

human EC, ES, and embryonic germ cells have also been shown to express a very high level of alkaline phosphatase.

Expression levels of alkaline phosphatase decrease following stem cell differentiation.

Human Embryonic Stem Cell Marker Antibody Panel (Catalog # SC008)

Each Kit Contains the following antibodies:

← Anti-Alkaline Phosphatase

← Anti-Nanog

← Anti-Oct-3/4

← Anti-SSEA-1

← Anti-SSEA-4

Human Embryonic Stem Cell Marker Antibody Panel Plus (Catalog # SC009)

Each Kit Contains the following antibodies:

← Anti-CD9

← Anti-E-Cadherin

← Anti-Nanog

← Anti-Oct-3/4

← Anti-Podocalyxin

← Anti-SOX2

← Anti-SSEA-1

← Anti-SSEA-4

A B C

An Embryoid Body Derived from Human Embryonic Stem Cells Stained for Pluripotency

Markers. Cells were stained using antibodies from the Human Embryonic Stem Cell Marker

Antibody Panel (Catalog # SC008) for Nanog (red; A) and the nuclei counterstained with

DAPI (blue; A) or for Oct-3/4 (green; B). Merge of images in (A) and (B) shows the overlap

of the 3 fluorochromes (white; C).

Images courtesy of Dr. Ronald McKay, NINDS.

Flow Cytometry analysis of SSEA-4 expression.

Reactivity of human embryonic stem cells stained

with mouse anti-SSEA-4 monoclonal antibody

(Catalog # MAB1435) (green) or isotype control

(black). Cells were stained using a PE-conjugated goat

anti-mouse IgG secondary antibody.

Primer Pairs for Embryonic Stem Cells

The Human and Mouse/Rat Pluripotent Stem Cell Assessment Primer Pair Panels profile the mRNA transcripts of fourteen

genes that are frequently used as markers for molecular characterization of germ cells, ectodermal, endodermal, and

mesodermal lineage-committed and undifferentiated embryonic stem cells. A primer pair for GAPDH is included and can be

used as a control for successful cDNA synthesis.

Pluripotent Stem Cell Assessment Primer Pair Panels

Primer pair panels have been specifically designed for Human (Catalog # SC012) and Mouse/Rat mRNA (Catalog # SC015).

Kit Contents:

← Primer Pair for GAPDH

← Positive Control

← Primer pairs for:

AFP HNF-3 beta/FoxA2 PDX1/IPF1

Brachyury Nanog SOX17

DPPA5/ESG1 Nestin SOX2

GAPDH Oct-3/4 Stella/DPPA3

GATA-4 Otx2 TP63/TP73L

Stem Cell Primer Pairs. PCR products derived from various tissue samples

were separated by agarose gel electrophoresis.

Undifferentiated ES Cells Ectodermal Lineage Endodermal Lineage Mesodermal Lineage Germ Cell

DPPA5/ESG1 Nestin AFP Brachyury Stella

Nanog Otx2 GATA-4

Oct-3/4 TP63 PDX-1

SOX2 SOX2 SOX17

HNF-3 beta

Neural Stem Cells

Primary Mouse Cortical Stem Cells

Read-to-use primary cortical stem cells isolated from E14.5 CD-1 mice.

Primary Rat Cortical Stem Cells

Ready-to-use primary cortical stem cells isolated from E14.5 Sprague-Dawley rats.

A Summary of Neural Stem Cell Molecules

Kits for the Neural Differentiation of Pluripotent Stem Cells

Kits to differentiate neural stem cells into dopaminergic neurons or oligodendrocytes.

Neural Stem Cell Proliferation Screening Kits

The Neural Precursor Cell-Based Screening & Bioassay Kit contains specially formulated media supplements to study the

proliferation and differentiation of neural precursor cells.

Neural Progenitor Cell Marker Kits

The Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel is designed for the identification and characterization

of human, mouse, or rat neural progenitor cells by marker expression using immunocytochemistry and flow cytometry

techniques.

StemXVivo™ Culture Matrix

A defined proprietary mixture of recombinant human adhesion molecules for the culture of stem/progenitor cells.

Cell Culture Media Supplements & Reagents

Additional supplies for cell culture.

Kits for the Neural Differentiation of Pluripotent Stem Cells

Human/Mouse Dopaminergic Neuron Differentiation Kit (Catalog # SC001B)

Background Information

The midbrain dopamine projections to the striatum have been among

the most extensively studied catecholamine neurons, in part because

the degeneration of dopaminergic (DA) neurons in the substantia nigra

results in Parkinson's disease. The basic organization of midbrain

dopamine neurons and their projections is consistent across most

mammals. Both animal models and clinical trials have suggested that

cell replacement therapies may be effective in treating Parkinson's disease. However, this approach is limited by the

availability of a rich, effective source of neural precursors for DA neuron generation.

Figure 1. Dopaminergic neurons generated using

Dopaminergic Neuron Differentiation Kit (Catalog #

SC001B). Cells were stained with Tyrosine Hydroxylase

(green), beta III Tubulin Tuj1 (red), and counterstained

with DAPI (blue).

Kit Description

The Human/Mouse Dopaminergic Neuron Differentiation Kit (Catalog # SC001B) is a system designed for in vitro

dopaminergic neuron differentiation of mouse ES cells in a serum-free environment. The kit contains ITS and N-2 Plus Media

Supplements, which are used to select and enrich neural stem cell populations. Bovine fibronectin is included to support

cell attachment and spreading. A growth factor panel, consisting of human fibroblast growth factor basic (FGF basic),

mouse fibroblast growth factor 8b (FGF-8b) and mouse sonic hedgehog amino-terminal peptide (Shh-N), is included for

effective dopaminergic differentiation. The quantity of each component provided in the kit is estimated to be sufficient for

induction of 3 x 107 embryonic stem cells.

Kit Contents

← FGF-8b

← FGF basic

← Shh-N

← Fibronectin

← ITS Media Supplement

← N-2 Plus Media Supplement

Mouse Oligodendrocyte Differentiation Kit (Catalog # SC004)

Background Information

Oligodendrocytes are myelinating cells in the central nervous system

(CNS) that form the myelin sheath of axons to support rapid nerve

conduction. In CNS disorders, such as stroke, multiple sclerosis and

spinal cord injury, demyelination of axons contributes to functional

deficit. Studies have demonstrated that enhanced remyelination of damaged CNS axons through transplantation can

restore functions lost as a consequence of demyelination. However, the approach of neural transplantation therapy is

limited by the availability of a rich, effective source of oligodendrocyte precursors for myelin regeneration.

Kit Description

The Mouse Oligodendrocyte Differentiation Kit (Catalog # SC004) is a kit designed for in vitro oligodendrocyte

differentiation of mouse embryonic stem cells in a serum-free environment. The kit contains ITS and N-2 Plus Media

Supplements, which are used to select and enrich neural precursor populations that are characterized by Nestin and A2B5

staining. Bovine fibronectin is included to support cell attachment and spreading. A growth factor panel, consisting of

human fibroblast growth factor basic (FGF basic), human epidermal growth factor (EGF), and human platelet-derived

growth factor AA (PDGF-AA) is included for effective oligodendrocyte differentiation. The quantity of each component

provided in the kit is estimated to be sufficient for the induction of 3 x 107 embryonic stem cells.

Mouse Oligodendrocytes. Oligodendrocyte lineage

cells were detected with mouse anti-oligodendrocyte

marker O4 (Catalog # MAB1326)

Kit Contents

← EGF

← FGF basic

← PDGF-AA

← Fibronectin

← ITS Media Supplement

← N-2 Plus Media Supplement

Neural Stem Cell Proliferation Screening Kits

The Neural Precursor Cell-Based Screening and Bioassay Kit contains specially

formulated media supplements to study the proliferation and differentiation of

neural precursor cells.

This microplate-based assay utilizes the redox sensitive dye, Resazurin, to

monitor cell proliferation. In parallel, differentiation of neural stem cells can be

monitored with HRP-conjugated mouse anti-neuron specific beta-III Tubulin

antibody. Reagents provided in the kit are sufficient for two 96-well plate

proliferation assays and two 96-well plate differentiation assays.

Neural Precursor Cell-Based Screening & Bioassay Kit (Catalog # SC014))

← Label: Resazurin (proliferation) and HRP-conjugated beta-III Tubulin (differentiation)

← Testing Format: Microplate reader

← Sample Type: Cultured cells

← Size: 4 x 96-well microplates*

*Each kit contains reagents for two proliferation assays and two differentiation assays.

Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel (Catalog # SC025)

The Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel is designed for the identification and characterization

of human, mouse, or rat neural progenitor cells by marker expression using immunocytochemistry and flow cytometry

techniques.

View Full Assay Principle

Kit Contents*

← Anti-CXCR4

← Anti-SSEA-1

← Anti-SOX1

← Anti-SOX2

← Anti-Musashi-1

← Anti-Notch-1

← Anti-Nestin

← Anti-Vimentin

*This kit requires secondary development reagents for immunocytochemistry, such as the NorthernLights™ Fluorescent Secondary Antibodies, and flow cytometry, such as anti-mouse IgG (Catalog # F0101B, F0102B, F0103B, or F0114) and anti-mouse IgM (Catalog # F0116, F0117, F0118, or F0119).

Detection of Neural Progenitor Cells using the Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel. A) Rat cortical stem

cells (Catalog # NSC001) or B) mouse cortical stem cells (Catalog # NSC002) were stained with anti-Nestin (A) or anti-Musashi-1 (B) antibodies

included in the Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel (Catalog # SC025). Cells in (A) were subsequently stained with

NorthernLights™ 557-conjugated donkey anti-mouse secondary antibody (Catalog # NL007; red). Cells in (B) were subsequently stained with

NorthernLights 493-conjugated donkey anti-goat secondary antibody (Catalog # NL003; green) and counterstained with DAPI (blue). C, D) Flow

cytometry was also used to detect rat cortical stem cells (C) or mouse cortical stem cells (D) stained with anti-SSEA-1 (C) or anti-CXCR4 (D)

antibodies (filled histograms) included in the Human/MouseRat Neural Progenitor Cell Marker Antibody Panel (Catalog # SC025). Open histograms

show staining with the appropriate isotype controls.