How are the billions of cells in our bodies made? In plants and animals, there are cells capable of producing every type of cell the organism will need from birth to death. Once an organism begins to mature, other cells are required to produce the range of specialized cells needed for a functioning organ or tissue. Such cells are known as stem cells. As technology has developed and the micro-dissection of tissue became possible, it was evident that most, if not all, organs and tissues in the body have their own stem cells which are capable of dividing and differentiating into mature functional cells. In some ways, stem cells can be imagined as blank canvases with many hidden cellular pictures already imprinted on them. Different combinations of proteins (growth factors) or other stimuli such as fats or sugars that touch the cell can stimulate division and allow the daughter cells to take on changed characteristics. In this chapter we look briefly at the types of stem cell and where they come from.

In simple terms, there are two major types of stem cells—embryonic and adult ( Figure　16 ). Embryonic stem cells have different biological properties to the adult stem cells that are found close to and after birth, hatching, or germination. The archetype of stem cells in development is the zygote, produced by the fusion of egg and sperm, which has the complete ‘potency’ to generate every tissue and cell type in the body (a property termed‘totipotent’). As the embryo grows, ‘pluripotent’ stem cells appear, which are limited in their potency. These cells divide and differentiate into the main classes of ES cells (known as the germ layers in mammals) and then, in turn, develop into organs and tissues. From embryo to adult, our cellular growth relies on: ‘multipotent’ cells that give rise, after division and differentiation, to cell types belonging to a related cell family; ‘oligopotent’ stem cells that are more limited still, producing just a handful of closely related cells, for example myeloid blood cells; and ‘unipotent’ stem cells that will give rise to only one cell type, for example muscle cells. The term progenitor or progenitor stem cell is often used to describe those cells still rapidly dividing but not as yet fully differentiated. Adult progenitor stem cells repair tissues by producing specific cell types needed to maintain normal turnover of regenerative organs.

16. The origins of embryonic and adult stem cells

Before we continue the story of stem cells, some developments in scientific technology need to be described as they have greatly accelerated our knowledge of this area. First, there was the serendipitous discovery of the presence of specific proteins and sugar molecules on the surface of various types of stem cells.

Antibodies to these molecules have accurately characterized stem cell populations. Secondly, a method called fluorescent activated cell sorting (FACS) uses fluorescently tagged antibodies, which allow living cells to be mechanically sorted according to their surface proteins. In simple terms, the suspension of cells is mixed with a particular fluorescent antibody and passed through a very narrow tube illuminated by a beam of laser light; a light-sensitive device detects those cells that produce a fluorescent glow. The positive cells can be analysed or collected from the mixture using electrostatic defiection of the fluorescent micro-droplets as they emerge from the tube. Using the same types of antibody but now attached to small magnetic beads, the partially purified stem cells can be isolated in sufficient number for clinical use. Cell populations can also be followed after transfusion into a recipient by attaching a small green fluorescent protein (GFP) gene to one of its own protein genes. Mutations of this jellyfish protein gene produce many different fluorescent colours (responding to light of specific wavelengths for accurate analysis). Whenever this gene is expressed, its protein fluoresces inside the cells. Similar ‘bioluminescence’ technology has been used to see individual cells within the living animal, often using the sensitive cameras originally developed for satellite surveillance.

Historical perspective

Ignored for two millennia, Aristotle, in his book　On the Generation of Animals , first proposed the theory of epigenesis in biology, suggesting that development of a plant or animal from an egg or spore follows a sequence of steps in which the organism changes and the various organs form. Though this theory now seems obvious in the genetic age, it was not given much credence because of the dominance of creationist and preformationist theories of life’s origins for many centuries. In 1795 the embryologist Caspar Friedrich Wolff famously refuted preformationism in favour of epigenesis. An extended and controversial debate by biologists finally led epigenesis to eclipse the long-established preformationist view. Visual understanding of cell populations continued with improvements in microscopy. At the turn of the 20th century, Ernst Neumann described the cells in the bone marrow and stated that

The different forms of all blood cells happening in the blood, the lymph-organs and in the bone marrow are all descendants of the ‘great-lymphocytic’ stem cell. In which way this stem cell completes itself again and again, whether exclusively by a mitotic division or also from other cells .

This was probably the first use of the term stem cell.

Embryonic stem cells

Stem cells found in the embryo can give rise to a large number of cells to generate all 200 of the cell types in the human body. In humans, the inner cell mass (see　Figure　17 ) of the early embryo has 50–150 cells of three primary types. In 1981, Martin Evans and Matthew Kaufman described a new technique for the culture of mouse embryos and the derivation of cultured embryonic cell lines. Later that year, Gail Martin first used the term ‘embryonic stem cell’ to describe these cell lines. Eight years later, James Thomson isolated a group of cells from the inner cell mass of the early human embryo and established the first embryonic pluripotent stem cell lines in culture. The current source of many human embryonic stem cells arises from　in vitro fertilization (IVF) procedures.

While sharing many similar biological properties, mouse and human embryonic stem cells (ES cells) require different environments for their sustained growth without differentiation in plastic flasks. For example, mouse ES cells grow on a layer of gelatin and only need the addition of the protein growth factor LIF, whereas similar human cell lines require a feeder layer of live mouse fibroblast cells and another growth factor (human fibroblast growth factor). Without optimal growth conditions, the cells stop dividing and rapidly differentiate. The growth of various ES cell lines has now been fine tuned and there is an increasing understanding of the genes involved in maintaining stem cell characteristics. The maintenance of pluripotency requires a regulatory network that ensures the suppression of genes that lead to differentiation. The default situation is to limit division and to differentiate. Nature has perhaps evolved an almost foolproof mechanism to protect the organism from the dangers of ES cells that evade the normal controls on growth.

Germ-line stem cells generate sperm or eggs (haploid gametes) which have half the normal number of chromosomes, and transmit genetic information from one generation to the next. Such cells are easily identified, retrieved, and manipulated in the fruit fly. In these flies, eggs develop on a string (or ovariole) within the fly ovary. At one end, a small number of germ-line stem cells move along at a predictable rate and differentiate into eggs within eight days. The stem cells are surrounded by three differentiated cell types—terminal filament cells, cap cells, and inner sheath cells—which help make up an anatomically simple tubular structure (germarium). The cells at the tip of the ovariole are organized into a niche that maintains and controls the germ-line stem cells. A special cell–cell junction is formed between the stem and cap cells. These junctions hold a germ-line stem cell at the anterior and prevent it from moving away where it might receive differentiation cues. A special signal protein is needed to maintain this junction and control the rate of germ-line stem cell division.

In plants, all stem cells are totipotent and are able to divide and differentiate into all the cell types needed to produce the entire organism. Totipotent was a term introduced by the Austrian botanist Gottlieb Haberlandt to describe a property known to all gardeners who have for millennia placed small parts of a plant such as leaves, stems, and roots into soil and water to procreate their precious stocks through cuttings. The first growth of an individual plant cell back into an entire plant (a carrot) was performed by Fred Steward in the late 1950s. Mouse-eared cress, Arabidopsis thaliana , is a much-studied small flowering plant useful for genetic studies. The growing tip is a completely undifferentiated tissue found in the buds that continues to make leaves, flowers, and branches throughout a plant’s life. The 30–40 stem cells that reside in this complex structure are surrounded by millions of differentiating cells, making this a complex model to study. Most geneticists have resorted to using root tips, which are less complex. By using a mutant that produces a high number of accessible shoot meristems (embryonic tissue), a gene expression map has been made of the meristem that has allowed the characterization and subsequent fluorescent marking of these elusive stem cells.

Adult stem cells

Historically, our understanding of stem cells began with observations of how adult stem cells generate vast numbers of mature daughter cells. Ernst Neumann’s far-sighted ideas in the area of blood production from the bone marrow demanded the physical culture of stem cells for the completion of the proof. This proof was not to arrive until 60 years later when researchers discovered various parts of this complex puzzle, showing that stem cells in the bone marrow could self renew as well as dividing and providing all the various mature cells in blood.

In 1961, Ernest McCulloch and James Till devised a series of experiments that involved injecting bone marrow cells into the tail veins of mice prevented from producing their own stem cells by a lethal dose of X-rays. Visible nodules were observed to grow in the spleens of the mice and these were in proportion to the number of bone marrow cells injected. McCulloch and Till speculated that each nodule arose from a single marrow cell, probably a stem cell. This animal-based measure of bone marrow stem cells provided the major tool for measuring stem cell numbers for the next 30years. In the early 1970s, Mike Dexter showed that it was possible to grow primitive bone marrow cells for many weeks in laboratory culture flasks if they had a stromal cell feeder layer (this is a varied collection of non-blood cell types present in the bone marrow).

In the decades that followed many more tissue-specific adult stem cells have been identified. These stem cells are all capable of division and differentiation into the component cell types of their respective tissue. The observed growth of new neurons in rats has suggested the existence of stem cells in the adult brain. This interesting observation was contrary to previous ideas that brain cells lasted a lifetime. Since then, stem cells have been demonstrated in the brains of adult mice, songbirds, and primates including humans. The growth of new neuronal cells (known as neurogenesis) is restricted to two locations in the brain. Neuronal stem cells can be grown in the laboratory as floating cell aggregates (neurospheres) that contain a large number of stem cells. By changing the culture conditions, they can be differentiated into both neurons (electrically excitable cells that process and transmit information) and glia (cells that feed and protect the brain’s neurons).

Other organs have stem cell populations that supply the mature cells required either continuously or at specific stages of development. Examples include breast stem cells that are the source of cells for the mammary gland during puberty. They have been isolated from both human and mouse tissue and, in culture, differentiate into luminal epithelial (the inner layer of potentially milk-producing cells) and myoepithelial cells (the outer layer) as well as having the ability to regenerate the entire organ in the mouse. Human olfactory adult stem cells can be harvested from mucosa cells in the lining of the nose. Rather like ES cells, they differentiate into a wide range of cell types and are seen as a potential therapeutic source because of the ease of harvesting, especially in older people. Hair follicles contain different types of stem cell and they can give rise to neurons (nerve cells), Schwann cells (which contribute to the myelin sheath), myofibroblasts (a cross between fibroblast and smooth muscle cells important in wound healing), chrondrocytes (cells that make and maintain cartilage), and melanocytes (melanin-producing cells that give you a sun tan). Basal cells make up about 30% of the epithelium of the lung and, in humans, are present throughout the airways. They are relatively undifferentiated and probably act as a stem cell for this tissue. Further understanding of their biological properties may lead to their use in lung regeneration. The process of assembling endothelial cells into the blood vessel lining mainly occurs during embryonic development. Initially, it was thought that these cells were derived from endothelial progenitor stem cells early in development, but in the 1990s putative adult endothelial stem cells were identified in adult mouse blood. Recent work suggests that adult endothelial progenitor cells are important in the production of blood vessels, especially when new blood vessel growth is required (and generated) by a growing tumour. Endothelial progenitor stem cells, like blood stem cells, are recruited into the blood stream by growth factors before homing to the tumour site. Destruction of these cells within the bone marrow can reduce the growth rate of a tumour, as no tumour can continue to grow larger than two millimetres in diameter without a blood supply.

Stem cell properties

Adult stem cell numbers remain as a constant small pool of cells within any given tissue. Given their propensity to divide, any escape from control could be fatal to the animal or plant. On the other hand, each cell needs to renew itself as well as supplying progenitor daughter cells capable of further rapid division and differentiation to generate the millions of cells needed for a functional organ or tissue. To explain this observation in bone marrow, Ray Schofield proposed the hypothesis that stem cells exist in a specialized site or niche. This niche is composed of a group of cells dedicated to the provision of a microenvironment for the maintenance of a single stem cell ( Figure　17 ) . Niches function as a ‘base camps’ in which stem cells are physically retained, acting as a lasting reservoir for tissue regeneration. By regulating the balance between self-renewal and differentiation, the niche plays an essential role in controlling stem cell fate and maintaining stem cell numbers. In most cases, adult stem cells retained in the niche sites are dormant until they receive signals from the microenvironment that stimulate stem cell division. What constitutes this signal still remains unclear. One daughter cell remains in the niche site as a stem cell while the other, which no longer fits in the niche, leaves and progresses through rapid division and differentiation to form mature cells. If the microenvironment is further stimulated, such as by applying a growth promoting protein, the rate of this process can be greatly accelerated. The niche model has been shown across a variety of stem cell studies in other systems such as in the ovaries of flies,plants, and in the colonic crypts of mammals. Whether this simple model is universal to all stem cells remains unclear.

17. A simple model of the stem cell niche. The cell in the niche is dormant until awakened by stimuli not yet fully understood. Following division, the niche accepts one daughter cell while the other, now a progenitor stem cell, divides many times and changes into millions of fully differentiated cells

Adult stem cells have the characteristic of plasticity or transdifferentiation which, in very simple terms, means that one stem cell type can, under different conditions, turn into another cell type. For example, in mice and men, embryonic, bone marrow, adult liver progenitor, and other stem cells can all produce mature cells of the liver. These changes can be performed in the laboratory by introducing growth factor proteins to the stem cells or by transplanting cells into the liver where they are able to repopulate and, in some cases, even improve liver function.

In the embryo there are three primary germ layer cell types: ectoderm (giving rise to the nervous system, tooth enamel, hair, and keratinocytes of the skin), endoderm (developing into the guts, respiratory system, and bladder), and mesoderm (responsible for bone, muscle, connective tissue, the middle layer of the skin, liver, and bone marrow ) . Surprisingly, all three of these cell types have been found to produce mature cells normally derived from a different lineage.

What is not clear is how this transdifferentiation of stem cells works. The fate of the stem cell is defined in part by its genetic profile at division but also by the external signals it receives. If hit by confi icting external signals, the cell can switch its genetic profile and change into a different cell type. Because they are relatively small and structurally indistinct, the visual identification of true adult stem cells has always proved difficult. It has been suggested that plasticity could be explained by the presence of two or more populations of stem cells within the tissue. For example, adult stem cells give rise to mature cell types, while a smaller number of germ stem cells are still able to produce all cell types. Another model that tries to explain the balance between self renewal of stem cells and those differentiating in the bone marrow, hair follicles, and the gut, proposes that a stem cell can exist in two different states. One is dormant, a stem cell retaining a full developmental potential, whereas the other stem cells are active, capable of producing large numbers of differentiated cells. The balance between inactive and active stem cells is controlled by the levels of various developmental signalling proteins which were first identified in fruit flies but now known to be vital to all animal cells.

Normal metabolic activities—especially DNA replication, when combined with environmental factors such as carcinogenic chemicals, UV light, and radiation—cause DNA damage. It is calculated that as many as one million individual molecular lesions per cell per day can occur. The cell has a collection of processes by which these damaged DNA components are identified and repaired. Most of these DNA changes are harmless, and some probably contribute to why we are all different. Some cause serious structural damage to the DNA molecule and alter the ability of the cell to survive. Examples of these lethal changes include the chemical cross-linking or breakage of the DNA strands. Failure of the normal processes of DNA repair is recognized in the cell by the protein p53 (mentioned in Chapter 3). The cell then either slips into a permanent state of dormancy known as senescence or dies by apoptosis. In the absence of a p53 response, the damaged cell may begin to undergo uncontrolled cell division resulting in a cancer.

All animal cells have an internal ageing clock. Every chromosome has protective structures at each end called telomeres, which are made up of repeats of the DNA sequence TTAGGG. Telomeres protect against the ends of chromosomes fusing, preventing the formation of ring chromosomes. Each time the cell divides, one or two copies of this sequence are not replaced. Eventually, after many divisions, this protection at telomeres begins to run out and the chromosome ends begin to ‘fray’, at which point division ceases. Embryonic stem cells avoid this limitation on the numbers of ‘permitted’ divisions by producing an enzyme called telomerase that repairs the damage and allows for the multiple divisions required in early development. In adult tissue, cells that need to divide continuously (for example immune cells and organ-specific stem cells) also have high levels of telomerase, whereas most other cell types express it at low levels. High levels of telomerase are often found in rapidly dividing tumour cells.

The stem cells at the growing tips of plants remain active throughout life, which may be centuries in some trees, during which time they are constantly exposed to environmental hazards that cause DNA damage and mutations. All plants have mechanisms that respond with great sensitivity to DNA damage, whether from radiation or cytotoxic chemicals, and signal an early cell death. Similar observations have been made in mouse bone marrow stem cells. The evolution of this stringent way of retaining their genomic integrity in these crucial cell populations is distinct from normal programmed cell death. On the other hand, there is an observed decline in the numbers of adult stem cells in many mammalian tissues with time, probably resulting from gradual low-level DNA damage. This affects their ability to divide with age by pushing cells into a dormant state. The number of stem cell niches may also decrease. We grow old because our stem cells age as a result of mechanisms that in our youth suppress the growth of cancer cells.

Cancer stem cells

The idea of cancer stem cells has slowly increased in credibility over the last ten years. The leukaemia stem cell was the first such cell to be described but it has been suggested that cancers of the brain, colon, ovary, pancreas, and prostate may also have stem cells. The origins of these cells are controversial. The concept is based on the notion that, as with normal stem cells, a pool of malignant cells has the ability to indefinitely self renew and re-initiate tumours in distant locations. As with normal stem cells, these cells only divide slowly (in other words self renew), which makes them more resistant to anti-cancer drugs that usually kill rapidly dividing cells, while their differentiated daughter cells remain sensitive. One explanation of leukaemia stem cells is that the genetic damage leading to cancer takes place in a newly differentiated cell followed by a de-differentiation event triggered by the stem cell niche, resulting in a very limited but immortal supply of leukaemic stem cells. In solid tumours it is extremely difficult to pinpoint the precise origin of the putative cancer stem cell as the tumour has a heterogeneous population of mutant cells. Among these cells there may be several types of stem cells, one optimal to the specific environment and several less successful lines. The latter cells can become more successful in some environments, allowing the tumour to further adapt to changes in its environment. Ongoing stem cell research may have repercussions on new cancer treatment.