Animal or plant cells, removed from tissues, will continue to grow if supplied with the appropriate nutrients and conditions. When carried out in a laboratory, the process is called Cell Culture. It occurs in vitro (‘in glass’) as opposed to in vivo (‘in life’). The culture process allows single cells to act as independent units, much like a microorganism such as a bacterium or fungus. The cells are capable of dividing, they increase in size and, in a batch culture, can continue to grow until limited by some culture variable such as nutrient depletion.

Cultures normally contain cells of one type although mixed cultures, especially of bacteria, are common in food sciences and wastewater treatment related studies. The cells in culture may be genetically identical (homogenous population) or may show some genetic variation (heterogeneous population). A homogenous population of cells derived from a single parental cell is called a clone. Therefore all cells within a clonal population are genetically identical.

Why Grow Cells in Culture?

There are a number of applications for animal cell cultures:

• To investigate the normal physiology or biochemistry of cells. For instance studies of cell metabolism.
• To test the effect of various chemical compounds or drugs on specific cell types (normal or cancerous cells, for example).
• To study the sequential or parallel combination of various cell types to generate artificial tissues e.g. artificial skin. Possibility of generating artificial tissues is an emerging and intensively studied area of biotechnology known as “tissue engineering”.
• To synthesize valuable biologicals from large scale cell cultures. The biologicals encompass a broad range of cell products and include specific proteins or viruses that require animal cells for propagation. For example, therapeutic proteins can be synthesized in large quantities by growing genetically engineered cells in large-scale cultures. The number of such commercially valuable biologicals has increased rapidly over the last decade and has led to the present widespread interest in animal cell culture technology.

The major advantage of using cell culture for any of the above applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells. The disadvantage is that, after a period of continuous growth, cell characteristics can change and may become quite different from those found in the starting population. Cells can also adapt to different culture environments (e.g. different nutrients, temperatures, salt concentrations etc.) by varying the activities of their enzymes.

Milestones of Cell Culture

The history of cell culture dates back to early twentieth century. The original impetus for the development of cell culture was to study, under the microscope, normal physiological events such as nerve development. The growth rate of animal cells is relatively slow compared with bacteria. Whereas bacteria can double every 30 minutes or so, animal cells require anywhere from 18 to 24 hr to double. This makes the animal culture vulnerable to contamination, as a small number of bacteria would soon outgrow a larger population of animal cells. Consequently, animal cell culture did not become a routine laboratory technique until the 1950s.

The need for cell culture, especially at large scales, became apparent with the need for viral vaccines. Major epidemics of polio in the 1940s and 1950s promoted a lot of effort to develop an effective vaccine. When it was shown in 1949 that poliovirus could be grown in cultures of human cells, considerable interest was shown to develop large quantities of the polio vaccine using cell culture. The polio vaccine, produced from de-activated virus, became one of the first commercial products of cultured animal cells.

Recombinant DNA technology (also known as genetic engineering) was developed in the 1970s to express mammalian genes in bacteria. It soon became apparent that large complex proteins (and especially those having therapeutic value) couldn’t be produced in bacteria, as they do not have the appropriate metabolism to add sugar chains to these proteins. Therefore, genetically engineered animal cells were developed for large-scale commercial production of such important proteins.

Another milestone in the animal cell culture technology came in 1975 with the production of hybrid cells (known as hybridoma) from the fusion of two or more cells capable of continuous production of a single type of antibody. These antibodies have diagnostic and therapeutic value and are now produced commercially in kilogram quantities from large-scale cultures of hybridomas.

Plant Cell Culture

Plant cells have been cultured to produce many ingredients needed by the food industries. Tremendous progress has been made in recent years in understanding the basics of plant metabolism and in the development of bioprocesses as well as design and operation of large-scale bioreactors for plant cell culture. A wide range of food ingredients including flavors, colorants, essential oils, sweeteners and antioxidants have been produced in culture. Japan has so far been the most successful country in the world to carry out plant cell culture on commercial scale. Ginseng products derived from cell suspension cultures of Panax ginseng used as an additive in wine, tonic drinks and herbal liquors have been produced by a company in Japan since 1990 with a net sale of 3 million dollars in 1995.

Types of Mammalian Cultures

Freshly isolated cultures from mammalian tissues are known as primary cultures until sub-cultured. At this stage, cells are usually heterogeneous but still closely represent the parent cell types as well as in the expression of tissue specific properties. After several sub-cultures onto fresh media, the cell line will either die out or ‘transform’ to become a continuous cell line. Such cell lines show many alterations from the primary cultures including change in morphology, chromosomal variation and increase in capacity to give rise to tumors in hosts with weak immune systems.

Animal cells can be grown either in an unattached suspension culture or attached to a solid surface. Suspension cultures have been successfully developed to quite large bioreactor volumes, with successful production of viruses and therapeutic proteins.

The range of commercially available biologicals produced by cell culture technologies has increased rapidly over the last decade. This is particularly so for therapeutic proteins synthesized from selected or genetically engineered mammalian cells. They are needed in large quantities and hence require careful study of the underling biochemical, cell biological and engineering principles for control of production processes.

Generally, this term refers to food crops that have been altered using a variety of molecular biology techniques in order to provide them with either new or enhanced characteristics. Examples of such enhancements of modifications are herbicide tolerance, pesticide resistance, greater nutritional content or increased tolerance of cold temperatures. Genetically modified organisms (GMOs) can also be referred to as transgenic organisms. Transgenic simply means that the organism’s genes come from more than one source.

The idea of enhancing desired traits in food crops is not new. Upon domestication of many plants, farmers used the process of artificial selection to grow plants with desired qualities. However this method can be time consuming and it is very difficult to introduce new traits into a specific population. In contrast, using genetic engineering, scientists can take the gene that controls the trait from one organism and insert it into another organism that does not have the gene. This creates an organism with the desired characteristic quickly and easily. A common example of genetic engineering is the insertion of Bacillus thuringiensis genes into corn to make Bt corn. Bacillus thuringiensis is a bacterium that naturally produces a protein that is lethal to insect larvae. By transferring the genes that encode this protein into corn, scientists have created a type of corn that produces its own pesticides, making it resistant to insects such as the European corn borer.

Transferring the gene

Taking a gene from one organism and inserting it into another is essentially a process of cutting the gene which codes for the trait of interest from the foreign organism and pasting this gene into the genome of the organism that you want to alter.

Let us use the insertion of B. thuringiensis genes into corn as an example. In order to cut out the gene of interest in the bacteria, its total DNA is isolated. Special enzymes, called restriction endonucleases, act as scissors to cut out the desired gene. These enzymes are sensitive to the DNA sequence and will only cut DNA at specific spots. There are many different enzymes that cut in different places, so the enzyme used depends on the sequence of DNA surrounding the desired gene.

Once the gene is cut out, scientists must make an “expression cassette.” This consists of additional DNA surrounding the gene so that the corn cell knows where the gene of interest begins and ends. The part that tells the corn cell where the gene begins is called the promoter and the end, the terminator. Once the expression cassette has been made, it is inserted into a plasmid. The plasmid is a parasitic circle of DNA present in bacteria. By putting the cassette into a plasmid, millions of copies of it can be made. These copies are then introduced into the host cell and get inserted into the genome. Cells which have successfully incorporated the foreign gene into their genome are then expanded in cell culture and used to generate new plants.

The ethics of GM Foods

GM foods have been the subject of much controversy. Advocates feel that GM foods will help provide food to the world’s continually expanding population. Since the number of people on earth keeps increasing (over 6 billion, and expected to double within 50 years), and the amount of land suitable for farming remains constant, more food must be grown in the same amount of space. Genetic engineering can make plants that will give farmers better yields through several different methods.

Crops can be harmed or destroyed by many different factors. Insects, weeds, disease, cold temperatures and drought can all adversely affect plants resulting in lower yields for the farmer. Genetic engineering techniques can be used to introduce genes, creating plants that are resistant or tolerant to these factors. Bt corn is an example of the introduction of a pest resistance gene. Monsanto has created strains of soybeans, corn, canola and cotton that are resistant to the weed-killer Roundup®. The weed-killer can be sprayed over the entire crop, killing all plants except the transgenic crop intended to be grown. Scientists have also taken a gene from a cold-water fish and introduced it into potatoes to protect the seedlings against sudden frost. These methods all create plants that are more likely to survive and be healthy, thereby increasing the production of farmer’s fields.

Genetic modification can also be used to change the properties of the crop, adding nutrients, making them taste better, or reducing the growing time. A good example of adding nutrients to food is the development of “golden” rice. Many countries in the world rely on rice as their primary food source. Unfortunately, rice is missing many essential vitamins and minerals, so people whose diet is based on rice are often malnourished. One of the most severe consequences of this is blindness caused by vitamin A deficiency. Researchers at the Swiss Federal Institute of Technology Institute for Plant Sciences genetically engineered rice, making it high in vitamin A. The group hoped to distribute the rice for free to any third world country requesting it.

Golden rice is a controversial subject in its own right. Its development was a breakthrough for biotechnology as it was the first time 3 genes were introduced simultaneously (generally, only one gene is transferred at a time). Mammals make vitamin A from beta-carotene, which is not found in polished white rice. A precursor to beta-carotene (geranyl geranyl diphosphate, or GGPP) is present, but three additional chemical reactions must be carried out to transform GGPP into beta-carotene. The gene transfer was successful, resulting in rice that is high in beta-carotene and is actually yellow coloured. On the surface, this seems like the solution to vitamin A deficiency.

Sounds great, right? GM foods can be grown easily, withstanding cold or drought, without spraying for pests or weeds. Not only that, but the food can be made more nutritious. So what’s the problem? Why so much controversy?

Opponents of genetic modification have many criticisms against this new technology. First of all there are multiple environmental concerns. GM foods can cause harm to other organisms unintentionally. For example, a study published in Nature on Bt corn found that the pollen caused high mortality rates in monarch butterfly caterpillars, even though the caterpillars don’t eat corn [1]. If the Bt corn pollen is blown onto neighbouring milkweed plants (the caterpillars food source) the caterpillars could eat the pollen and die. The results of this study are under debate, since the experiments were not done in the field, but in a laboratory, and new studies suggest that the original may be flawed. Researchers at the University of Guelph performed a study and found that under natural conditions, Bt corn does not pose a risk to the monarch butterfly [2].

Similarly, if pollen is blown onto neighbouring plants, the plants could crossbreed and the introduced gene could be transferred to non-target plants. This is a concern if a herbicide resistant crop were to breed with a weed and transfer the herbicide resistance gene. This would create a weed that is unharmed by the chemicals used to kill it.

Monsanto has patented their Roundup Ready seeds, and farmers wishing to use them must purchase a license from the company. This can lead to trouble for farmers who don’t use the Monsanto seeds. Perry Schmeiser is a canola farmer in western Canada who has never bought seeds from Monsanto. In 1998 he was sued by Monsanto since they discovered Roundup Ready canola in his field. Schmeiser claims that the seed was blown in from neighbouring fields, but Monsanto believes he obtained it illegally or stole it. Regardless of how it was obtained, Monsanto felt this was patent infringement and took Schmeiser to court in June of 2000. This court battle captured the interest of farmers around the world, because even if they did not intend or even want to have patented seeds in their fields, they could be sued. The judge ruled in favour of Monsanto and stated that it didn’t matter how the seed got into Schmeiser’s field. Whether it was blown in, cross-pollinated by birds, bees or animals, fell off farmer’s trucks or migrated from a neighbour’s field, it is still patent infringement, and the plants were to become the property of Monsanto. All of Schmeiser’s profits from 1998 were awarded to Monsanto since there was a probability of having the genetically altered seeds throughout his fields.

Insect pests may also become resistant to the toxins produced by GM crops like Bt corn. It is now known that some bacteria are becoming antibiotic resistant (so-called “superbugs”) making it difficult to treat diseases such as tuberculosis. Likewise, opponents of GMOs believe that insects could become pesticide resistant making them difficult to control in the future.

Along with environmental concerns, there are also worries about the effects that GM foods can have on humans. There are concerns that introducing a new gene into a food could cause an allergic reaction in some people (for example, if the gene came from a nut). Most scientists believe that other than allergic reactions, GM foods do not pose a threat to human health, however as with all new products, no long-term studies have been performed.

How are genetically modified foods regulated in Canada?

The Canadian Food Inspection Agency (CFIA) is responsible for the control of GM foods in Canada. The CFIA has strict criteria that must be met before a GM food can be marketed. These include: how the food crop was developed, including the molecular biological data which characterizes the genetic change; composition of the novel food compared to non-modified counterpart foods; nutritional information for the novel food compared to non-modified counterparts; potential for new toxins; and potential for causing an allergic reaction. Once the government is satisfied that requirements have been met, the food is approved for consumers. Right now, Canada has no mandatory labeling policy for GM foods, it is strictly on a volunteer basis, however mandatory labeling is required if the introduced gene poses an allergy risk (eg. if the gene introduced came from a nut) or if the food’s nutritional content has changed. A Canadian standard for labeling of biotechnology derived foods is being developed and is expected to be completed in the fall of 2002.

The CFIA has approved 51 “novel foods”, most of which are GM foods, including corn, (types resistant to corn borers and herbicides); canola, (varieties resistant to herbicides); potato (varieties resistant to Colorado potato beetles); tomato (varieties that ripen slowly); squash; soybean; sugarbeet; flax; and cottonseed oil.

References

1. Losey JE, Rayor LS, Carter ME. Transgenic pollen harms monarch larvae. Nature 399, 214 (1999).

2. Sears MK, Hellmich RL, Stanley-Horn DE, Oberhauser KS, Pleasants JM, Mattila HR, Siegfried BD, Dively GP. Impact of Bt corn pollen on monarch butterfly populations: A risk assessment. Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):11937-42.

(Art by Jen Philpott)

Biotechnology is the application of scientific and engineering principles to process biological materials for goods and services. Biotechnology is, therefore, highly multidisciplinary with foundations in many fields including cell and molecular biology, physiology, immunology, microbiology, genetics, biochemistry and chemical and process engineering.

Biotechnologists uses techniques derived from a variety of disciplines (fig. 1). Their main objectives are the innovation, development and optimal operation of processes in which biochemical catalysis plays a fundamental role. Biotechnology relies on each contributing discipline to better understand the technical language, potential and limitations of other areas.

Historically, biotechnology was an art rather than a science, exemplified in the manufacture of products like wine, beer and cheese. Manufacturing techniques were usually discovered by chance but then thoroughly and reproducibly worked out. However the molecular mechanisms were not understood. With major advances in biochemistry and microbiology, these processes have become better understood and improved. Modern biotechnological processes now include a wide range of new products including antibiotics, vaccines and antibodies and a variety of therapeutic proteins. Biotechnology has been further diversified by many new molecular innovations, allowing unprecedented changes to be made to living systems. Transgenic plants and animals are igniting a new era in agriculture and human gene therapy promises to eradicate many diseases which are currently incapacitating and untreatable. Environmentally, biotechnology is allowing major improvements in water and land management as well as bringing solutions to pollution generated by various industries.

A key factor distinguishing a traditional biologist from a biotechnologist is the scale of operation. The biologist usually works in the range of nanograms to milligrams. Biotechnologists, depending on the project, may aim to generate quantities of desired product in kilograms or higher. As such, biotechnology aims to amplify biological processes.

The developments in biotechnology are currently proceeding at a speed similar to that of microelectronics in the 1970s. Although the analogy is tempting, it is probably not realistic to expect that biotechnology will develop commercially at the same spectacular rate experienced by microelectronics. Nonetheless, biotechnology will still have a considerable impact across all industrial uses of the life sciences, in spite of the fact that some traditional means of production are still economically more favourable than the newer biotechnological methods. Chemical means of production that utilize petrochemical based feedstocks are still more economically sound compared to biotechnology driven routes for most industrial chemicals. Biotechnology will undoubtedly have great benefits in the long run in all sectors.

Many new biotechnology companies have spun off from universities. These companies are often technologically driven and multidisciplinary in nature. Product development can involve molecular biologists, clinical researchers, bioprocess engineers and sales staff. The business climate of biotechnology companies is often characterized by rapid change and considerable risk as one biotechnology innovation may quickly supercede another. Another peculiar feature of new biotechnology companies is that their business growth is often highly dependent on venture capital, as they require exceptionally high level of funding before profit sales return.

Biotechnology & Chemical Engineering

Many biotechnological processes may be considered as having a three-component central core, in which one part is concerned with obtaining the best biological catalyst for a specific function or process, the second part creates (by means of technical operation) the best possible environment for the catalyst to perform, and the third part (downstream processing) is concerned with the separation and purification of an essential product or products from a fermentation process.

In the majority of processes so far developed, the most effective, stable and convenient form for the catalyst has been the whole organism. It is for this reason that so much of biotechnology revolves around microbial processes. However, this does not exclude the use of higher organisms, in particular plant and animal cell culture that have played a vital role in the development of therapeutic and diagnostic biological products and will continue to play an increasingly important role in biotechnology.

The second part of the core of biotechnology deals with all aspects of the containment system or bioreactor within which the catalysts must function. Here the combined knowledge of the scientist and the bioprocess engineer interact, providing the design and instrumentation for the maintenance and control of physiochemical environment such as temperature, aeration, pH, etc., thus allowing the optimum expression of the biological properties of the catalyst.

The third aspect of the biotechnology, namely downstream processing, can be a technically difficult and expensive procedure. Downstream processing is primarily concerned with the initial separation of the bioreactor medium into a liquid phase and a solid phase, and subsequent separation, concentration and purification of the product. Chemical engineering principles play a vital role here as well in terms of designing and operation of the separation systems. Downstream processing costs can be as high as 60 – 70% of the selling price of the product as exemplified by the plant Eli Lilly built to produce human insulin. Over 90% of the 200 staff are involved in the recovery processes. Downstream processing represents a major part of the overall cost of most processes but is also the least glamorous aspect of biotechnology. Improvements in downstream processing will benefit the overall efficiency and cost of processes and will make the biotechnology competitive to the conventional chemical processes.

Biotechnology and the Environment

Biotechnology has been successfully employed to reduce or eliminate various forms of air and water pollutants. Methods developed by environmental biotechnologists might use the microorganisms to either break down or sequester pollutants. The concept of using microorganisms to treat pollution problems is not a new idea; microbes were first used as early as 1930s to treat industrial wastewaters. More recently they helped in the cleanup of oil spilled from the Exxon Valdez tanker off the coast of Alaska in 1989 as well as decontaminated water aquifers contaminated with various industrial chemicals such as phenol, trichloroethylene (TCE) and compounds present in various petroleum fractions.

Opportunities for using microorganisms for bioremediation of soils contaminated with various industrial pollutants arose when scientists discovered that there is practically nothing that is not viewed as food by one microbe or another. Just as some insects can feed on leaves that are toxic to others, so some microbes can thrive on molecules that would poison most organisms. Microbes exist in nature that feed on toxic materials such as methylene chloride, detergents, phenol, sulfur and polychlorinated biphenyls (PCBs).

Microbes metabolize the toxic compounds to produce harmless end products such as carbon dioxide, water and salt. The chemical conversions usually involve breaking large molecules into several smaller molecules, much as we break down the complex carbohydrates in our food to simple sugars such as glucose. In some cases, the by-products of a bacterial banquet are not simply harmless but actually useful. Methane, for example, can be derived from a form of bacteria that degrades sulfite liquor, a waste product of paper manufacturing. Methane can then be used in a wide range of industrial applications such as electricity production

Although individual species of bacteria can carry out several different steps of chemical breakdown, most toxic compounds are degraded by group of bacteria living together. Each species in the group work on a particular stage of the degradation process and all of them together are needed for complete detoxification.

Depending on the nature and extent of pollution, microbial clean up can take from a few weeks to a year or more. When the toxic chemicals are gone, the population of bacteria itself dwindles, being replaced by other microbes native to the area and more suited to the new conditions.

(Art by Jane Wang – note that high res versions of image files available here)

Stem cells have generated more excitement, scrutiny and controversy than any other area of recent scientific study. The first stem cells, which were discovered half a century ago, were isolated from blood. Now, scientists around the globe are researching various types of stem cells for their potential to regenerate lost tissue and revolutionize medicine.

Embryonic stem (ES) cells are derived from the embryo when it exists as a blastocyst. They have the ability to develop into all the different cell types found in the body. Actually, when a sperm fertilizes an egg, the resulting single cell begins to divide and multiply at a rate much faster than that observed in somatic cells. Scientists refer to these cells as totipotent stem cells (Figure 1). These primordial embryonic cells have the potential to grow into a complete mammal. Within days of fertilization, these new and dividing cells form a hollow sphere, called a blastocyst. Stem cells arising in the inner mass of the blastocyst are called the ES cells. The ES cells are considered pluripotent – they can divide indefinitely and blossom into all the various tissue types of the human body, but they have the lost the totipotent ability to grow into a separate being. After roughly 14 days, ES cells divide to give rise to what will eventually develop into the spine. At this stage, the stem cells within the embryo are considered multipotent. These stem cells can grow into some tissues, but not all tissues. Those destined to become bone or blood, for example, may not be able to form stomach or skin.

By definition, stem cells have two important characteristics that distinguish them from other types of cells. First, they are unspecialized cells that renew themselves for long periods of time through cell division of at least one daughter cell. Secondly, under certain physiological or experimental conditions, they can be induced to differentiate. This means that they can divide into cells with special functions, such as the beating cells of the heart muscle or the insulin-producing cells of the pancreas.

Discovery of ES Cells

The work that laid the foundation for ES cell discovery was the study of teratocarcinomas, complex tumors containing a mix of specialized cell types as well as a population of unspecialized cells. These unspecialized cells are called embryonal carcinoma (EC) cells. The latter were shown to be pluripotent and could give rise to various cell types both in vitro and in vivo. It was therefore natural to consider using these cells for therapeutic purposes. However, EC cells never seemed ideal for this purpose because they had an abnormal number of chromosomes and originated from tumors. Careful study of the induction of teratocarcinomas in experimental animals, as well as an understanding of the biology of EC cells and early embryos, led scientists to the discovery of ES cells in the early 1980s. The demonstration that ES cells contained the normal number of chromosomes and were truly pluripotential has influenced many scientific disciplines.

Biological role and properties of 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; they can choose to become one of the many different types of cells present in the body based on signals from their environments.

Finding the answers to two fundamental questions about stem cells that relate to their long-term self-renewal is crucial to our ability to successfully grow these cells in laboratory and in turn use them for various tissue engineering and cellular therapies. The first question deals with why embryonic stem cells can proliferate for extended periods of time in the laboratory without adopting a specialized fate, while most adult stem cells cannot. The second issue addresses which factors in living organisms normally regulate stem cell self-renewal and differentiation. 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 ultimately leads to cancer.

Stem cells are unspecialized

One of the fundamental properties of stem cells is that they do not perform specialized functions. A stem cell cannot pump blood through the body (like a heart muscle cell) or supply oxygen to the cells of the body (like a red blood cell). However, unspecialized stem cells can give rise to specialized cells including heart muscle cells, blood cells, or nerve cells. They do this by coordinating their gene expression in an elaborate and complex pattern spanning many generations of cells.

Stem cells are self- renewing

Most specialized cells in our body (such as muscle cells, blood cells, or nerve cells) do not replicate themselves. The supply of these cells is maintained by stem cells, which replicate many times and then differentiate into the specialized cells that are needed. In this way, cells are continuously replenished as they die. This is called homeostasis.

When cells replicate themselves many times over it is called proliferation. A starting population of stem cells that proliferates for many months in the laboratory can yield hundreds of millions of cells. If the resulting cells are identical (in terms of being unspecialized themselves and capable of generating specialized cells) to the parent stem cells, the ES cells are said to be capable of long-term self-renewal. Scientists are greatly interested in determining specific factors and conditions that allow stem cells to remain unspecialized in the laboratory for long periods of time.

Stem cells can give rise to specialized cells

Differentiation is the process whereby stem cells give rise to specialized cells. Scientists are just beginning to understand the signals that trigger stem cell differentiation. These signals include chemicals secreted by other cells, physical contact with neighboring cells, and contact with molecules in the microenvironment.

Can specific sets of signals be identified as responsible for the promotion of differentiation into specific cell types? Answering this question is critical in order to learn how to maintain pools of stem cells. The development of stem cell-based technologies will depend on our ability to reproducibly drive the differentiation of stem cells into specific tissue lineages. This issue is of particular importance for the development of ES cell-based technologies, where the differentiation of certain subsets of cells and tissues is uncommon. The generation of ES cell-derived heart muscle cells is one example where significant improvements in tissue-specific differentiation are needed. Under default mechanisms, only approximately 1% of ES cells form cardiac tissue.

Growing embryonic stem cells in the laboratory

The process of growing cells in the laboratory is known as cell culture. Mouse (or human) embryonic stem cells are isolated by transferring the inner cell mass of the blastocyst into a plastic laboratory culture dish containing medium, which provides nutrients and necessary growth factors. The cells divide and spread over the surface of the dish. In the case of human ES cells, the inner surface of the culture dish is typically coated with mouse embryonic skin cells, which are non-dividing and secrete a host of growth factors. This is known as a feeder layer and is necessary for the prevention of ES cell differentiation. The feeder layer provides a sticky surface to which the inner cell mass cells can attach. Mouse ES cells, however, can be maintained in culture without the need of a feeder layer. In this case, the culture medium is supplemented with high concentrations of a protein known as leukemia inhibitory factor (LIF), which prevents differentiation.

Over the next several days, the cells of the inner cell mass grow and divide and spread on the culture dish. When the cell concentration is high enough, they are removed gently and plated into several fresh culture dishes. This process of replating the cells is repeated for many months, and is called subculturing. After six months or more, the original 20 to 30 cells of the inner cell mass yield many millions of embryonic stem cells. When embryonic stem cells have proliferated in cell culture for six or more months without differentiating, are pluripotent, and appear to be genetically normal, they are referred to as an ES cell line. Once ES cell lines are established, batches of them can be frozen and kept for further experiments or even shared with other laboratories for further culture and experimentation.

ES Cell Differentiation

As long as mouse ES cells in culture are grown in the presence of LIF or a feeder layer, they can remain undifferentiated (unspecialized). If LIF is removed from the culture medium, cells will begin to clump together to form 3-dimensional structures called embryoid bodies (EBs). The cells then begin to differentiate spontaneously. Allowing spontaneous differentiation is a good way to determine if a culture of embryonic stem cells is healthy. It is not an efficient way to produce cultures of a specific cell type. In order to generate a large number of cells of a specific type, scientists use the process of directed differentiation. This is done by changing the chemical composition of the culture medium, altering the surface of the culture dish, or modifying the cells by inserting specific genes.

Adult Stem Cells

Many adult tissues (such as the bone marrow, brain and gut) contain stem cells. Like ES cells, adult stem cells can make identical copies of themselves for long periods of time (self-renewal). At the same time, they can give rise to mature cell types that have characteristic shapes and specialized functions. Stem cells typically generate an intermediate cell type(s) before they achieve their fully differentiated state. The intermediate cell is called a progenitor cell. Progenitor cells are partly differentiated cells in the sense that they are committed to a particular cell lineage and, upon division, and give rise to differentiated cells. Of all the adult stem cells identified thus far, Hematopoeitic stem cells (HSCs) are the best characterized.

Stem cell plasticity

Until recently, adult stem cells were considered to be irreversibly committed to specific lineages of differentiation. HSCs, for example, normally give rise to all types of blood cells such as red blood cells, white blood cells and platelets. It was previously believed that HSCs could not give rise to any cells of a different tissue. However, a number of experiments over the last several years have raised the possibility that stem cells from one tissue may be able to give rise to cell types of a completely different tissue. This phenomenon is known as ‘stem cell plasticity’. Examples of such plasticity include bone marrow stem cells becoming neurons, or pancreatic islet cells that are capable of producing insulin. Exploring the possibility of using adult stem cells for cellular therapies has recently become an active area of research. The apparent plasticity of adult stem cells has forced scientists to reconsider many fundamental concepts of stem cell biology. Many new questions arise from these studies. For instance, are the recently characterized stem cells typical tissue-specific stem cells? Might they be a sub-population of stem cells with a developmental potential closer to that of embryonic stem cells? There is a possibility that stem cell plasticity results from in vitro manipulations and does not reflect normal behavior in vivo. Regardless, this phenomenon has important clinical implications.

Potential Therapeutic Applications of Stem Cells

The ultimate objective of stem cell bioengineering is to be able to understand and possibly control stem cell differentiation and lineage commitment in vitro. If this can be achieved, a multitude of therapeutic applications can be envisioned. One potential application is the generation of different types of neurons for the treatment of Alzheimer’s disease, spinal cord injuries, or Parkinson’s disease. The production of heart muscle cells for heart attack survivors may also be possible. The generation of insulin-secreting pancreatic islet cells for the treatment of type-1 diabetes, and even the generation of hair follicle stem cells for the treatment of certain types of baldness, have been considered. Stem cells could also be useful for a number of tissue engineering applications such as the production of complete organs including livers, kidneys, eyes, hearts, or even parts of the brain. This represents a considerably greater challenge, beyond the generation of specialized cell types, and will require considerable time and effort to develop. Other areas that would benefit from a better understanding and control of stem cell proliferation in vitro are drug testing, cancer research, and fundamental research on embryonic development.

Human ES cells could potentially be used for all of the above applications. Human ES cells are obtained from aborted fetuses or fertilized eggs. This has come under ethical scrutiny since use of these procedures requires serious moral consideration by society. A possible way to circumvent this issue would be to use stem cells isolated from adult tissues. If, as recent studies have suggested, the plasticity of stem cells is greater than previously imagined, then it may be feasible to use adult stem cells as an initial cell population for therapeutic modality. Bone marrow, peripheral blood or umbilical cord blood HSCs could be ideal for such procedures. They are relatively easy to harvest and robust assays exist to quantify HSCs and distinguish them from more differentiated progenitors. Although much remains to be learned about the factors controlling their self-renewal and differentiation, the state of knowledge about HSCs is considerably more advanced than for other stem cell systems.

(Art by Jen Philpott)