Most diseases have cellular origins. The majority can be traced to simple biochemical imbalances that can often be corrected by drugs. The cells return to a near-normal state and the patient feels better. Some complex diseases start with gene changes that prevent a particular protein being expressed or change the protein structure in such a way that its function is altered or ineffective. Even with the checks and balances of internal repair or cell suicide, some gene changes escape and, when involved in the fundamental cell processes, they can have a devastating effect on cell growth or function. Increased growth caused by gene changes leads to the slippery slope of cancer where these ‘out of control’ cells further change their genetics and develop the ability for limitless division and, in some cases, gain the ability to invade other tissues. In simple terms, treating such diseases requires that the errant cells are removed, corrected, or even better replaced. Many other diseases are not directly life threatening but cannot be corrected by drug treatment because of their complex biochemical nature. The ideal solution would be to treat the disease at its root by replacing the faulty cells. This process is called cellular therapy and can be divided into different forms. The most familiar is the transplantation of mature functional cells, as in a blood transfusion, and stem cells as in bone marrow transplants. We still await a standard therapy for the introduction of modified human or animal cells that replace a required substance, such as insulin-producing cells for diabetics.

Many quote the Swiss physician and alchemist Philippus Aureolus Paracelsus as the first person to describe the concept of cell therapy in his book D er grossen Wundartzney (‘Great Surgery Book’) published in 1536 in which he wrote: ‘the heart heals the heart, lung heals the lung, spleen heals the spleen; like cures like’. This remark came from his theory that eating healthy animal organs would rebuild and revitalize the particular ageing or faulty organ—more of a nutritionist doctrine than one of a modern cellular therapist. As long ago as 1667, Jean-Baptiste Denis, working in the royal laboratory of Louis XIV, attempted to transfuse blood from a calf into a mentally ill patient. The first recorded non-blood transfusion occurred in 1912 when German physicians tried to treat children suffering from under-active thyroid with transplanted thyroid cells. In 1931, a Swiss clinician Paul Niehans became the ‘father of cell therapy’ by accident when he introduced minced bovine thyroid tissue into a rapidly deteriorating patient who had experienced severe damage to his thyroid glands during surgery. The patient recovered and lived for a further 30 years. Niehans became renowned for his cellular treatments, and his patients included members of various Royal families, Pope Pius XII, politicians, and famous film stars. His name and some treatments, especially skin and beauty processes, live on at the Paul Niehans Clinic founded in Switzerland by his daughter. The 20th century is littered with stories of so-called cellular therapies. John Brinkley, known as the ‘goat gland doctor’, reportedly did 16,000 operations in which he implanted men with tissue from the testicles of young goats, asserting that this procedure was effective against impotence and could cure conditions ranging from acne to insanity. His licence was revoked on the grounds of immorality and unprofessional conduct. James Wilson promoted the use of bovine connective tissue cells. He claimed that such cellular preparations taken by mouth ‘have the ability to migrate to any tissue in need of repair and, once at the site, take on the characteristics of the healthy cell it associates with’. Other attempts along similar lines caused hundreds of deaths by violent immune reactions to bacterial or viral infections including two men who died of gas gangrene following injections of foetal sheep cells. Other, even more questionable embryonic cell transplant therapies were pioneered by Niehans in Switzerland. In the 1970s, his student Wolfram Kuhnau set up a clinic in Mexico, where there were no regulations, using embryonic blue shark cells purchased off local fishermen and with a dubious number of live cells, to ‘treat’ patients for a wide range of diseases. As a result of this ‘quackery’ and the bad press it received, it is no wonder that there was a high degree of scepticism about cell-based therapies as we approached the 21st century.

Transplantation of blood cells

Blood transfusion using animal blood started in the late 15th century but was unsuccessful because of blood group incompatibility and infections. Such was the death toll that transfusion was banned throughout Europe for over 150 years. Around the turn of the 20th century, Karl Landsteiner discovered human blood groups (A, B, AB, O), for which he received the Nobel Prize in 1930. Mixing blood from individuals with incompatible groups leads to blood clumping or agglutination. Landsteiner discovered that this process was due to an immunological reaction between dissimilar blood groups. The classification of blood groups is based on the presence or absence of inherited antigens, including proteins, carbohydrates, and lipids, on the surface of the red blood cells. If incompatible blood is introduced, antibodies in the recipient’s blood plasma attack the new red blood cells and destroy them in a reaction called haemolysis, resulting in renal failure and circulatory shock. Blood transfusion is now a routine procedure and most blood donations are fractionated into their components. These include red blood cells, platelets, white blood cells, plasma, and various proteins such as antibodies and clotting factors (see Chapter 5). Selected components allow for specificity of treatment, reduction of side effects, and efficient use of a unit of blood. For example, platelets can help restore the blood’s clotting ability when given to people with too few platelets (thrombocytopenia), a condition leading to severe and spontaneous bleeding and which is sometimes a side effect of chemotherapy. While the transfusion of white cells is rare for treating infections, it has been used in therapy when the cells have been genetically manipulated to elicit any anti-tumour cell activity.

In the 1970s, Edward Donnall Thomas demonstrated that bone marrow cells infused intravenously can repopulate the bone marrow and produce normal blood cells, work for which he received the Nobel Prize in 1990. The active cells in these repopulating preparations are adult blood stem cells. Cytotoxic drugs required for cancer treatment destroy all dividing cells and consequently do not discriminate between tumour cells and blood stem cells. Replacing the patient’s stem cells following chemotherapy replenishes the bone marrow cell population and promotes the resumption of normal blood cell production. Bone marrow transplantation has been used in a wide range of cancer therapies including leukaemia and lymphoma to repair bone marrow damage caused by chemotherapy. Bone marrow transplants have also been successful in treating cases of anaemia and other diseases where stem cells are damaged or absent.

Autologous blood stem cell therapy uses the patient’s own stem cells, which are removed and purified prior to treatment. The advantages of autologous transplants are that they are relatively free of infection and the recovery of immune function is more rapid and increased. Autologous transplants are not always possible and so other sources of stem cells are needed. Allogeneic blood stem cell transplants involve a healthy donor whose tissues must be a close tissue match to those of the patient. The closer the donor (often a relative) is genetically to the patient, the better the match of specific cell-surface proteins, called the histocompatibility complex. Even a single DNA base pair difference resulting in a changed amino acid sequence of one of the five histocompatibility proteins will result in a mismatch. Leading bone marrow transplant centres are able to sequence the DNA of all five histocompatibility genes to check for compatibility. Only identical twins give perfectly matched stem cells but unrelated donors need as many matches as possible. Mismatching increases the risk of graft rejection or graft versus host disease. Graft rejection occurs when the body rejects the new transplanted cells while graft versus host disease occurs when the new cells reject the body. Both effects result in an immunological reaction that can be fatal.

In early treatments, bone marrow was taken from a large bone of the donor, typically the pelvis, through a needle that reaches the centre of the bone. The technique is referred to as a bone marrow harvest and is performed under general anaesthesia with all the requirements of hospitalizing the donor. Nowadays stem cells can be sourced from circulating blood. This method was developed from an observation that circulating stem cells increased dramatically following injections of a blood protein growth factor.Donors are given growth factor and their stem cells are collected by a mechanical separator, after which the red cells are returned to the donor.

It is also possible to isolate useful amounts of blood stem cells from amniotic fluid and the umbilical cord. Cord blood has a high concentration of stem cells, but only enough for blood stem cell transplants in young children. Using combinations of growth factors, it is possible to amplify the numbers of stem cells in the umbilical cord allowing possible use in adult transplants. Umbilical stem cells generally produce low levels of graft versus host disease. Storage of one’s own umbilical cord stem cells for possible future use in adult life has developed into an expanding business for tissue storage companies.

Organ stem cell therapy

In the early 1960s, mouse embryonic carcinoma cell lines having stem cell characteristics were derived from a teratocarcinoma. This cancer tissue is made up of germ cells that are derived from the ovary directly or indirectly due to birth defects resulting from errors during embryo development. The use of these cells was hindered by problems of genetic mutations and genome instability. The isolation of ES cell lines from normal embryos (see Chapter 6) overcame these defects and began a new area of research, exploring the possibilities of isolating and manipulating human embryonic cells for possible use as stem cell treatments in adults.

Mouse ES cells have been used extensively in the generation of transgenic mice. These genetically modified mice are extremely useful, as they act as a model to study the functions of individual genes in a species close to humans. Transgenic mice are created by transferring a gene (for example a human cancer-related gene) into cultured ES cells. Alternatively, individual genes can be ‘knocked out’ by introducing a foreign gene—often a drug resistance protein—into a target gene. Unfortunately these gene manipulations are notoriously inefficient, and consequently the treated cells need to be grown in culture and selected for the correct gene change. The selected cells are then micro-injected into the inner cell mass of a normal embryo before implantation into the uterus of a foster mother mouse. The offspring have a copy of the introduced gene present in one of their paired chromosomes (i.e. they are heterozygous) and can be further bred to gain homozygous strains in which the new gene (or knocked out gene) has a copy in both matched chromosomes, so the effect will be apparent in every cell in the animal. Whether the gene is expressed in a particular tissue or cell type will depend on its genomic environment, something that can usually be directed at the ES cell level by careful molecular selection. There are now thousands of transgenic mice strains each with a specific gene alteration and they have greatly enhanced our understanding of many complex biological processes. When an error or the over expression of a particular gene is the major cause of a disease, transgenic animals can act as a model for developing new drugs or pharmaceutical interventions. Even when these genetic manipulations introduce changes that result in aborted foetuses, cell lines can be ‘rescued’ and grown in culture for useful research.

There are a number of possible sources of human ES cells: (i) cadaver foetal tissue, (ii) embryos remaining after infertility treatments, (iii) embryos made solely for research purposes using IVF, and (iv) embryos made using nuclear transfer into eggs (the technique used to create Dolly the sheep). All human ES cell sources raise ethical and religious questions. Country by country, political regulation usually refiects the predominant ethical and religious views, with many countries completely prohibiting all work on embryos, others having strict regulations, while some have few, if any, restrictions. Opponents of human embryo stem cell research believe that a human life begins as soon as an egg is fertilized. The destruction of an embryo is deemed morally as murder. They also argue that ES cell technologies are a first step to reproductive cloning, which fundamentally violates and devalues the sanctity of life. Proponents argue that in the natural reproductive process, human eggs are often fertilized but fail to implant in the uterus. A fertilized egg, while it may be capable of forming human life, cannot be considered a human being until it has been successfully implanted in a woman’s uterus. The methods required for IVF routinely create more human embryos than are needed over the course of a fertility treatment, leaving excess embryos that are often simply discarded and it is morally permissible to use such embryos for potentially life-saving biomedical research. ES cell lines from early 1990s experiments have been allowed limited use in clinical treatments.

One example of using ES cells to treat human injury comes from the approval in 2009 for the first phase of clinical trials for transplantation of human stem cells of the brain and spinal cord(oligodendrocyte progenitor cells), derived in culture from human ES cells, into patients with injured spinal cords. The first patient was treated in October 2010 by Hans Keirstead’s team at the University of California, Irvine and sponsored by the biotechnology company Geron. Experiments in rats had previously shown that there had been improvement in the recovery of movement in animals with spinal cord injuries after a seven-day delayed transplantation of human ES cells that had been forced into oligodendrocyte lineage in culture. This new ongoing study to treat paraplegics will last for at least five years. While it is not expected that this treatment will completely cure the injury, it is hoped that it will show sufficient repair of the nerve cells to be worthwhile. This is a pioneering study promising a bright future for the use of ES cells for treating spinal injuries.

The ethical, religious, and political constraints of isolating embryonic cells for human therapy has driven the research dream, and now the reality of inducing or de-differentiating adult cells into embryo-like pluripotent cells that could be used in cellular therapy. In 2006, Shinya Yamanaka produced induced pluripotent cells from mouse fibroblast cells by forcing the expression of several genes. Retroviruses (a DNA virus family that inserts its sequence into the host DNA) were used to induce the expression of genes identified as important for maintaining ES cells in the adult fibroblast cells. Early attempts did not prove totally successful as the expression of some viral genes caused cancer after transplantation into the mouse embryo. This problem was overcome in 2008 by using an adenovirus to introduce genes. Unlike retroviruses, this virus does not incorporate its own genes into the host cell genome. Several groups mirrored these mouse experiments in adult human cells. In the following year, Sheng Ding and colleagues found it was possible to change somatic cells into pluripotent cells without oncogene insertion but using repeated treatment with two small chemically synthesized proteins. The manipulation of an individual’s adult cells to produce embryonic-like cells capable of repairing their organs and tissues is predicted to be a major clinical treatment of the future.

The potential of using stem cells to repair diseased and damaged organs without the risk of organ rejection or other side effects is an endpoint that is driving much research. Stem cell therapies exist but, so far, only as experimental medical treatments. The main areas of progress are in the treating of cardiac and muscle damage, diabetes, liver, Parkinson’s disease, and Huntingdon’s disease.

Cardiovascular disease—including hypertension, coronary heart disease, stroke, and congestive heart failure—is the major cause of death in many countries around the world. When deprived of oxygen, cardiac muscle cells (cardiomyocytes) die and this triggers the formation of scar tissue, the overload of blood flow and pressure, and the over-stretching of viable cardiac cells leading to heart failure and death. Using mouse, rat, and pig models, various types of stem cells including embryonic, mesenchymal, endothelial, and naturally occurring heart stem cells have been shown to regenerate damaged heart tissues. A few studies in humans, usually undergoing heart surgery, have shown that stem cells introduced directly to the heart or transfused into the blood circulation have given improved cardiofunction and induced the formation of new capillaries.

Muscular dystrophy is a group of genetic disorders in males that causes the muscles to weaken with time and eventually leads to premature death. The condition is caused by changes in the protein dystrophin which normally maintains the integrity of muscle. Using mouse and dog models, stem cells called mesoangioblasts (programmed to differentiate into muscle cells) with a corrected dystrophin gene have been transplanted. Normal dystrophin levels and muscle strength were regained in four out of six dogs, suggesting that cellular therapy may be the way ahead for treatment of this genetic disease.

In 2008, ES cells were coaxed into developing into immature beta cells capable of producing insulin, which were able to reverse diabetes in mice. In the same year, adult human skin cells, first induced into pluripotent cells, were also reprogrammed to produce insulin. A more exciting aspect was that these cells secreted insulin in response to glucose (as they do normally in the cells within the pancreas). It is only a matter of time before transplantation of insulin-producing cells will replace the need for a lifetime spent injecting insulin in people suffering from type 1 diabetes. Similar progress has been made culturing liver cells from induced adult stem cells in mice, making liver regeneration a distinct possibility.

The gradual loss of dopamine-producing nerve cells in certain areas of the brain results in Parkinson’s disease. Early cellular treatments relied on transplanted foetal brain tissue. A few individuals showed a marked improvement and provided a general proof of principle. Apart from the contentious tissue source, these clinical trials did highlight several issues including the need for large quantities of pure cells and that the foetal transplants became affected themselves by transmission of Parkinson’s disease. In 2008, induced progenitor stem cells were created from skin fibroblasts of mice and then further differentiated into neuronal precursor cells which (following transplantation) integrated into the surrounding brain. This approach has been further extended in an animal model which mimics Parkinson’s disease by killing off the normal dopamine-producing cells with a toxin. Transplantation of induced stem cells gave a marked improvement compared to non-treated animals. The major challenge is now to understand how Parkinson’s disease develops and to use stem cell models to develop new drug treatments.

Huntington’s disease is a neurodegenerative disease that is characterized by the death of neuron cells of the brain that produce a neurotransmitter chemical (gamma aminobutyric acid). Clinical treatment using stem cells for Huntington’s has mirrored that of Parkinson’s disease. Foetal tissue and induced adult stem cells grafted into the adult brain increase brain activity, including motor and cognitive functions. Stem cells may not be the ‘magic bullet’ for this disease but could form a major part of its effective management.

Mesenchymal stem cells, found in the bone marrow, can be induced to form cartilage, bone, tendon and ligaments, muscle, skin, fat, and nerve cells. They are easy to isolate from small quantities of bone marrow and are readily grown to the amounts needed for transplantation. Frozen stocks are viable and could be used for ‘off the shelf’ therapies. They are also found in dental pulp, the soft tissue inside teeth. Wisdom teeth are a particularly rich source of stem cells and this opens up the interesting possibility of visiting the dentist for a stem cell transplant. Embryonic mesenchymal stem cells have been transplanted into the jaw bone of adult mice. Some of these ‘tooth germs’ grew into fully functional hard teeth capable of responding to pain. Whether adult tooth-derived stem cells can be persuaded to produce regenerated teeth remains uncertain but an Indian biotechnology company has set up the world’s first dental stem cell bank.