PROTEASE INHIBITOR COCKTAILS
Proteases are destructive molecules that break down proteins by catabolizing peptide bonds, turning proteins into amino acid chains or smaller peptides. Proteases are found endogenously in practically all organisms, and are used in natural biological processes such as blood-clotting, metabolism of proteins, and the regulation of apoptosis.

Invasive proteases can contaminate research samples and slow or halt research progress, rendering time-consuming and expensive work useless. Protease inhibitor cocktails are a unique series of enzyme inhibitors that knock out specific proteases to avoid peptide bond hydrolysis and subsequent protein destruction. They are used widely in primary research and in drug development.

Protease inhibitors can be classified by the particular protease they inhibit, or by their mechanism of action. For example, protease inhibitors can inhibit cysteine, serine, threonine, aspartic, or matealloproteases, meaning they prevent degradation of that protease’s substrate. The most common mechanisms of action of protease inhibitors are suicide inhibition and transition state inhibition, although there are others. Suicide inhibitors, often used in classical drug design, bind irreversibly and site-specifically to a protease enzyme, rendering it inactive. Transition state inhibitors are those that mimic transition states, binding to the substrate during a transition state and preventing the protease from cleaving or otherwise degrading that substrate. AG Scientific maintains a large selection of standard inhibitor cocktails in stock, as well as the ability to prepare custom formulations.




NECROSIS
Necrosis is the premature death of cells in living tissue and can be caused by external factors to the cell or tissue, such as infection, toxins, cancer, infarction, poisons, ROS (Reactive Oxygen Species), inflammation or trauma.

Historically, cell death has  been subdivided into regulated (apoptosis, aka programmed cell death) and unregulated (necrosis) forms. While apoptosis has always been recognized to be a pathway of highly coordinated signaling events which is a naturally occurring cause of cellular death and can often provide beneficial effects to the organism.

Necrosis is morphologically characterized by a gain in cell volume (oncosis), swelling of organelles, plasma membrane rupture and subsequent loss of intracellular contents. Necrosis or necrotic cell death is almost always detrimental and can be fatal. Currently, necrotic pathways are poorly defined and are still largely identified in negative terms by the absence of apoptotic or autophagic markers.Biochemically it is characterized by loss of regulation in ion homeostasis, random digestion of DNA & ultimately postlytic of DNA fragmentation. Physiologically, necrosis affects groups of contiguous cells, phagocytosis by macrophages and significant inflammatory immune response

Enclosed is  our line of novel Neucrotic & Necroptotic reagents to provide the tools you require to advance your research.

NECROPTOSIS
Newly research is determining that necrosis is not just a series of unregulated, uncontrollable processes but is in fact a series of  'programmed necrosis' aptly named necroptosis. 

Historically, Necrosis has been considered an accidental cell death and not set to determined pathways or cellular regulation. Necrotic cell death is defined by an increase in cell volume, swelling of organelles, plasma membrane rupture and eventual leakage of intracellular components. Now it is becoming increasingly realized, Neurotic cell death may be executed through defined mechanisms or pathways aptly termed "Necroptosis" (Degterev et al 2005). In fact, it may be executed via stimulation of tumor necrosis factor alpha (TNFα), FasL and TRAIL. These are the same ligands that activate APOPTOSIS.

Thus, cell death induced by the activation of the death receptor may be accomplished through alternative death pathways or necroptosis. Receptor interacting protein (RIP) kinases constitute a family of seven members, all of which contain a kinase domain (KD). They are crucial regulators of cell survival and death. Specifically, RIP1 kinase activation is required as an upstream regulator of necroptotic death pathway.

Additionally,  after TNFα stimulation identified Cylindromatosis (CYLD), a tumor suppressor, as an important regulator for mediating both Apoptosis & Necroptosis. It appears ubiquitination/deubiquination may be involved in the signal transduction of both Apoptosis & Necroptosis. Preliminary research demonstrates the inhibition of CYLS results in the suppression of Necroptosis by up-regulating wnt signaling. The wnt signaling pathway is a network of proteins best known for their roles in embryogenesis, cancer, basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division.

Necroptosis downstream mechanisms are still very preliminary and require further elucidation. Reactive oxygen species (ROS) is shown to be the executioner of necroptosis from some cell types and research has determined antioxidant treatment does not rescue all cell types from necroptosis. 

Mitochondrion are well defined in apoptotic cell death although there is preliminary mitochondrial involvement in necroptosis. Specifically, there is a downstream role for RIP1, adenine nucleotide translocase (ANT) and cyclophilin D (cypD) in the mitochondrial permeability transition has been proposed for necroptosis. In a cardiac ischemia experiment, a reduction in necrotic cell death occurred in cypD-deficient mice (Nakagawa et al. 2005).

Finally, autophagic vesicles are frequently observed in necroptotic cells thus the possibility of autophagy as an execution mechanism for necroptosis. It appear initial thoughts of autophagic cell death could actually be enlighten to demonstrate it is actually necroptosis.


APOPTOSIS
Apoptosis is the process of programmed cell death. It is a normal and necessary phenomenon for many biological developmental processes, allowing cells to differentiate from each other and to form proper connections. It is also a critical phenomenon in aging and in healthy cell turnover; the healthy adult human body sheds tens of billions of cells each day in order to maintain homeostasis. Excessive or faulty apoptosis is associated with many human disorders such as neurodegenerative and auto-immune diseases and the proliferation of cancers. The machinery and signaling pathways that control cell life and death are key areas in human therapeutic research, the manipulation of which offer great potential to alter the course of many human diseases.

CANCER RESEARCH
Cancer is most accurately a class of diseases characterized by uncontrolled cell growth or division. There are more than 100 types of cancer that can occur in nearly every organ or tissue in the body. The risk of cancer increases with age and also with certain genetic predispositions and/or exposure to carcinogens. Various types of cancer have become more treatable as research efforts continue towards eradicating these diseases. Research into the treatment of cancer has flourished particularly in the past decade, recently involving biotechnological tactics such as immunotherapy and gene therapy. Current therapeutic targets for the treatment of cancers include surface proteins of malignant cells as wells as heat shock proteins and other molecules involved in signaling pathways for processes critical to cancer growth such as angiogenesis, cell differentiation, proliferation, and growth.

MOLECULAR BIOLOGY
The fascinating field of molecular biology aims to understand the relationships between and regulation of different systems in a cell. Molecular biology is closely tied to the fields of biochemistry and genetics, and these fields share many techniques, approaches, and imminent goals. Molecular biology procedures are powerful tools used to copy DNA, identify which molecules are present in a sample, discover when and why a particular gene will be expressed, and more. Applications of molecular biology extend from biophysics to evolution, virology to neuroscience, and computation/modeling to cancer.

CELL CYCLE
Most of the human cell cycle is spent in the interphase. This is the period in which the cell grows, replicates its DNA, and acquires nutrients needed for the rest of the cell cycle, which is known as mitosis. Upon entering mitosis, the cell divides itself into two identical cells, and each new cell enters an independent interphase. The intricate regulatory processes of the cell cycle allow healthy cells to divide and force unhealthy cells to be repaired or to undergo apoptosis. These regulatory processes are the subject of intense scrutiny for application into the treatment of human disease, particularly a variety of cancers.


NEUROCHEMISTRY
The field of neurochemistry investigates the functionality of chemicals in the brain and nervous system. Neurotransmitters, possibly the most widely-known neurochemicals, are the chemical basis of communication between cells in the nervous system. Other neurochemicals include a milieu of proteins and lipids that work together to make up the r’s and other dementias, addiction, spinal cord injury, and neurodegeneratioalluring aggregate of human conscious and unconscious functions. Current applications of neurochemical research aim to understand and improve prognoses in diseases such as Alzheimen.

ANGIOGENESIS
Angiogenesis is the ability to form new blood vessels. This natural process is critical to development of blood flow architecture in utero and to wound healing throughout life. The body regulates angiogenesis by maintaining a balance of growth and inhibitory factors.

Improper blood vessel growth is linked to several human diseases including cataracts and age-related blindness, skin diseases, stroke and cardiovascular disease, and cancer proliferation. When cancerous cells in the middle of a tumor are low in oxygen, they can send angiogenic signals to the exterior of the tumor to indicate the need for more blood flow. In this case, tumors can develop their own blood supply, allowing a tumor to acquire more oxygen and nutrients and thereby to grow and spread.

Some newer cancer therapies involve blocking angiogenesis in order to restrict cancerous cells from thriving. Conversely, angiogenesis stimulation is used as therapy to speed the process of wound healing, enhance areas with weak circulation, and may prove effective in stimulating growth in damaged nerves and tissues such as the brain and heart.

 
PROTEASES & PHOSPHATASES
Proteases and phosphatases break down bonds via hydrolysis. Proteases break down peptide bonds of proteins into amino acid chains or smaller peptides. These are found endogenously in practically all organisms, and are used in natural biological processes such as blood-clotting, metabolism of proteins, and the regulation of apoptosis. Medical applications of various types of proteases range from treatment of ischemic stroke via manipulation of blood-clotting to anti-inflammatory functionalities.

Phosphatases remove phosphate groups from monophosphate esters, leaving a free hydroxyl group. These are found in practically all tissues, body fluids, and cells, and are used in natural biological processes such as bone calcification and metabolism of carbohydrates.  play an important role in many signal transduction pathways, as the addition or removal of a phosphate group can activate or de-activate an enzyme, or allow a particular protein-protein interaction. Medical applications of various types of phosphatases range from regulation of gene expression to diagnostic testing for a variety of disorders including anemia, liver function, and various types of cancers.

Individual proteases and phosphatases have specific laboratory and clinical functionalities; A.G. Scientific offers a wide variety of these enzymes to cater to your research, development, or processing needs.

IONOPHORES/ION CHANNELS
Ionophores are molecules that facilitate ion passage in or out of cell membranes. They can do this by binding to particular ions and acting as a mobile carrier, escorting them through the hydrophobic environment of cell membranes, or they can form ion channels. Ion channels form pores in membranes through which ions can pass. There are several types of ion channels, each regulated by various mechanisms designed to allow only certain ions to flow into and out of a cell and only at certain times.

Ion channels are named by their ion selectivity and by their opening/closing mechanism, which is also known as gating. Voltage-gated and ligand-gated ion channels are the most abundant and well-studied gating mechanisms, but there are also light-gated channels, mechanosensitive channels, second messenger channels, and others. Although there are some non-selective ion channels, most ion channels are selective for particular ions, allowing only ions of particular size and charge to pass through.

All endogenously present ions pass in and out of cell membranes via ion channels including calcium, potassium, sodium, chloride, and hydrogen protons. In the human body ionophores are closely connected with functions ranging from digestion to mental health. Ionophores are used for diagnostic radioimaging, they are components of many pharmaceuticals, and are used widely in research to increase or decrease ion concentration in solution.

UBIQUITIN-PROTEASOME PATHWAY
The ubiquitin-proteasome pathway is broadly linked to the regulation of nearly every cellular process. It is the main mechanism for catabolism of proteins, which breaks down large protein molecules so the cell can use parts to produce energy, recycle parts to make new molecules, or excrete parts that are no longer needed. The ubiquitin-proteasome pathway involves two major processes: conjugation and degradation. Conjugation is the process of targeting a substrate protein whereby several ubiquitin molecules attach to it, and degradation is the breakdown of that substrate protein by proteasomes. Certain types of ubiquitin molecules may also be responsible for processes such as DNA repair and endocytosis. The ubiquitin-proteasome pathway is closely tied to the functionalities of the cell cycle, gene expression, immune and/or stress response, apoptosis, and many others.

GENE SELECTION
Methods and applications of gene selection are constantly improving, as is the basic science leading to a more complete understanding of this process and its functions. Genome analyses reveal insight into evolutionary development of humans and other species. Answers to developmental and pathological questions are being answered by correlating gene expression to particular conditions. By examining the activity levels of thousands of genes, researchers are beginning to reveal which genes are responsible for the molecular events that are involved in particular diseases. Gene selection can also be experimentally modified with lab reagents such as selection antibiotics. Such studies are leading to therapeutics, diagnostic tools, and even predictive technologies for a wide array of diseases.

CELL AND TISSUE CULTURES
Cell cultures comprise the nutritious environment for cultivating cells. Particular nutrients can be used to dictate cell fate in processes including maturation, gene selection, and even cell form and function. The same is true for tissue cultures. Cell and tissue culture protocols can help determine the nutrients to use for nearly every type of cell cultivation or manipulation commonly used to date. Different nutrients are required for optimum harvest, isolation, or preservation of cells and tissues. A.G. Scientific offers a wide variety of standard media and culture products, as well as the ability to provide custom culture products or media quickly upon request.

BIOLOGICAL DETERGENTS
Detergents cleanse hydrophobic molecules by nature of their amphiphilic properties. Biological detergents are used to purify, isolate or solubilize membrane proteins while preserving the protein’s biological activity; they are also used to selectively prepare culture media by inhibiting certain bacteria growth, and to isolate, purify, crystallize or renature proteins. A high-quality biological detergent with the right properties can increase your yield and lower your costs. It is important to use a detergent of the highest quality, as some can be contaminated with undesirable oxidizing compounds. For your protein solubilizing and protein purification needs, A.G. Scientific carries the highest quality zwitterionic CHAPS and MOPS buffer, sulphobetaines, and dust-free SDS.

CYTOSKELETON
Proteins in the cytoplasm that provide structure to a cell are collectively known as the cytoskeleton. In addition to providing structure and protection to individual cells by acting as its musculature and skeleton, the cytoskeleton is involved in a cell’s mobility, division, organization, import and export.

Three categories of proteins comprise the cytoskeleton, which is present in all cells. These are actin filaments, intermediate filaments, and microtubules; each has distinct structure, properties, and roles in cytoskeletal functioning. With the action of motor proteins, the cytoskeleton is involved in nearly every contraction, motion, and intracellular exchange that takes place inside a cell or on its surface.

GROWTH FACTORS
Growth factors are a wide family of molecules that affect cell growth, proliferation, or differentiation. Different types of growth factors reside in different tissues and fluids of the body, performing various individualized functions from nerve regeneration to blood vessel differentiation. Some growth factors, such as BDNF are thought to be involved in the pathology of mental disorders such as schizophrenia and the perception of pain. Other growth factors such as insulin-like growth factor-1 and epidermal growth factor are implicated in various types of tumors and cancers. Many growth factors are currently being investigated and utilized for their therapeutic potential.

QUALITY IS OUR PRIORITY
A.G. Scientific is committed to the highest quality standards of biochemicals. We provide care and commitment to each project entrusted to us, at every step of the way from order processing to packaging and shipping. We ensure accuracy, consistency and accountability by way of a detailed documentation trail and friendly, accessible customer service.

We continually strive to be a globally-recognized competitive, full-service supplier of biochemical and laboratory reagents by utilizing trained employees and state of the art technology to provide our customers with quality components. We give your project the individual attention and care it deserves, from research laboratory to development to processing and any step between.

A.G. Scientific, Inc. is ISO 9001:2008 certified. This means that every chemical that goes through our hands is traceable to the highest quality guarantee. We comply with the highest regulatory standards of quality management, and we run our company not only according to honest and efficient business practices but also in a constantly improving manner. We pride ourselves on continually improving our overall efficiency and our ability to respond to customer needs and expectations.

 
ISO 9001:2008 certification requires a company to create and maintain a successful quality system framework. We believe we go beyond these requirements. The organizational processes making up the A.G. Scientific, Inc. quality management system comprise mutually beneficial end-to-end activities that allow our company to consistently complete projects on time and within budget.

Our clients are secure knowing their projects are being handled in a comprehensive, competent and confidential manner. Meticulous standard operating procedures are tailored to our individual client’s projects and extensive records are kept for every step of a project including:

• Manufacturing
• Testing
• Packaging
• Labeling
• Shipping

A.G. Scientific, Inc. achieves our continued quality goals by maintaining a quality management system that focuses on continual improvement through teamwork, individual responsibility, personnel development, and open communications with our employees, customers, and suppliers. This quality management system is executed and continually improved with the genuine purpose of supplying our clients with chemicals and reagents of the highest quality, usefulness, and innovation. 

CUSTOM FORMULATION/PRIVATE LABELING SERVICE
A.G. Scientific, Inc. provides comprehensive packaging and labeling services. We can cut your product’s handling and labor costs while applying the highest quality standards, resulting in hands-down improvement in your project’s efficiency and profitability.

Our packaging and labeling capabilities include:

• Custom or private labeling
• Custom cardboard packaging
• Custom product inserts
• Stringent tolerances and in-process volume checks to ensure accuracy
• Precision equipment for accurate fills
• Liquid fills (0.5ml to multi-liter)
• Powder fills (mg to multi-kilogram)
• Sterile and non-sterile packaging
• Crimp seal
• Stopper
• Screw cap
• Tamer seals
• Shrink-wrap
• Humidity control
• Safety canisters with vermiculite
• Contract lyophilization for small to large runs
• Inert gas overlay
• Flexible to your special handling requirements
• Capable, amenable and highly dedicated staff

A.G. SCIENTIFIC BACKGROUND
A.G. Scientific, Inc. was founded in 1997 in San Diego, CA with the purpose of Accelerating Scientific Discovery^TM. We strive to produce the most innovative and highest quality biochemicals available to the scientific community. The scientists we are privileged to have as customers are making significant advancements in molecular biology, neurobiology, cell biology, cancer research, drug discovery, agricultural biotechnology and animal health.

AG Scientific has grown to provide starting materials for Active Pharmaceutical Ingredient (API) manufacture, critical diagnostic chemicals, and medical device reagents. A.G. Scientific has demonstrated particular success in producing and supplying novel fermentation and naturally derived products including enzyme inhibitors, gene selection antibiotics, and reagents for protein purification.

We are very proud to be part of this larger effort to improve medicine and the well-being of our society as a whole. Our clients can be confident that their projects are being handled in a comprehensive and confidential manner. Contact us with your specific need and let us dedicate our expertise to your project.

SAN DIEGO SCIENTIFIC COMMUNITY
A.G. Scientific, Inc. is centralized amidst the biotechnology hotbed of San Diego. The city is home to prestigious research facilities such as The Salk Institute and The Scripps Research Institute and to a wealth of entrepreneurial and end-market companies. San Diego also boasts some of the country’s finest biotechnology research and education at institutions such as University of California, San Diego, San Diego State University, and University of San Diego. San Diego is one of the nation’s leading research locations and in previous years has been awarded the third largest dollar amount of health research funding in the country, behind Boston and New York.

A wealth of biotechnology industry has grown from research and education in the San Diego Area, and the area now boasts the third largest concentration of biotechnology companies in the world, behind Boston and the Bay Area.  The life science community in Southern California comprises more than 1,800 companies and employs more than 105,000 people. In 2007, Southern California was the recipient of nearly $1.8 billion in research funding from the National Institute of Health and nearly $1.7 billion in healthcare venture capital, according to San Diego Association of Governments. The life science industry in San Diego carries an annual economic impact of nearly $9 billion. Many cooperative public-private and academic-industrial partnerships exist in the San Diego area, allowing the scientific community to thrive in  this region.

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.

1. Stem cells are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity.
2. Stem cells, 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:

1. Embryonic Stem Cells: Scientists discovered ways to derive embryonic stem cells from early mouse embryos nearly 30 years ago, in 1981. In 1998 this research of mouse stem cells led to 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.
2. Non-embryonic "somatic" or "adult" stem cells: 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 called induced pluripotent stem cells (iPSCs).

Stem cells are important for living organisms for numerous 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 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.

Stem Cell’s unique regenerative abilities offer new avenues for treating diseases such as diabetes, and heart disease. However, significant preclinical research remains on how to apply clinical applications for cell-based therapies to treat disease, (aka regenerative or reparative medicine). Research on stem cells continues to advance knowledge on stem cells essential properties, what makes stem cells different from specialized cell types, how an organism develops from a singlecell and how healthy cells replace damaged cells in adult organisms.

What are the unique properties of all stem cells?

Stem cells differ from other kinds of cells in the body with have three general properties:

1. 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, stem cells can replicate many times and if the resulting cells continue to be unspecialized,  the cells are said to be capable of long-term self-renewal. Questions remain in the our  understanding two fundamental properties of stem cells that relate to their long-term self-renewal: such as why can embryonic stem cells proliferate for a year or more in the laboratory without differentiating, but most non-embryonic stem cells cannot.,What are the factors in living organisms that normally regulate stem cell proliferation and self-renewal?  Understanding  normal & abnormal  embryonic development may provide enlightenment into when cellular division leads to cancer. The specific factors and conditions that allow stem cells to remain unspecialized are of great interest to scientists. Following the development of conditions for growing mouse stem cells, twenty years elapsed until scientists learned how to grow human embryonic stem cells in the laboratory.
2. 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.
3. Stem Cells can give rise to specialized cell types. 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. Initial understanding of the intracellular & extracellular signals 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.
   1.  For example, are the internal and external signals for cell differentiation similar for all kinds of stem cells?
   2. Can specific sets of signals be identified that promote differentiation into specific cell types?
   3. c.      End goal:  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.

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.

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  use several kinds of tests, including:

1. Growing and subculturing the stem cells for many months. This ensures that the cells are capable of long-term growth and self-renewal.
2. Scientists inspect the cultures through a microscope to see that the cells look healthy and remain undifferentiated.
3. Test  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.
4. Tests  to determine the presence of particular cell surface markers that are typically produced by undifferentiated cells.
5. 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.
6. Determining whether the cells can be re-grown, or sub-cultured, after freezing, thawing, and re-plating.
7. 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?

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.

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 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 increasing the chance of compatibility 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?

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

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.

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.

Primary Source: The National Institutes of Health Resource for Stem Cell Research