STEM CELLS AND TISSUE RENEWAL One cannot contemplate the organization of tissues without wondering how these astonishingly patterned structures are established and how they are maintained over time, even as the cells of which they are made ultimately perish and are replaced. Although the structure of an animal’s body may be enormously complex, it is generated by a limited repertoire of cell activities—examples of which have been described throughout this book. Cells grow, divide, migrate, interact with other cells, and die. They generate forces and form mechanical attachments that organize tissues into organs (see Figure 20–25). They differentiate into specialized cell types by switching on or off specific sets of genes. They produce molecular signals to influence neighboring and distant cells, and they respond to signals that other cells deliver to them. They remember the effects of previous signals they have received, and so progressively become more and more specialized in the characteristics they adopt and the functions they carry out. These processes are launched at fertilization, when they guide the development of an organism from a fertilized egg. But they continue throughout an organism’s life, generating the cells that are needed to allow each tissue to operate smoothly, recover from injury, and—when possible—compensate for age-related decline. In this section, we review how adult tissues are organized and maintained. We examine the developmentally flexible cells, called stem cells, that drive the remarkable process of tissue renewal. Lastly, we describe techniques that allow researchers to generate cells that possess stem cell–like properties and can be coaxed into forming a specific tissue type—a feat that holds promise for the study and treatment of human disease. A Fertilized Egg Gives Rise to Every Cell Type and Tissue in the Body During the process of development, the fertilized egg cell divides repeatedly to produce a clone of cells—about 30,000,000,000,000 for a typical human. This clonal collection may take the form of a daisy or an oak tree, a hummingbird or a whale, a sea urchin or a mouse (Figure 20–32). No one builds these impressive structural displays: they self-assemble during development. Some visual examples of the initial steps in this magnificent undertaking are presented in Movie 20.3, Movie 20.4, and Movie 1.1 (see Chapter 1), which display the earliest developmental stages of a fruit fly, a zebrafish, and a frog, respectively. Figure 20–32 The genome of the fertilized egg determines the ultimate structure of the clone of cells that will develop from it. (A and B) A sea-urchin egg gives rise to a sea urchin; (C and D) a mouse egg gives rise to a mouse. (A, courtesy of David McClay; B, courtesy of the Alaska Department of Fish and Game; C, courtesy of Patricia Calarco. D, US Department of Agriculture, Agricultural Research Service.) The ability to produce a fully formed, complex multicellular organism— a property called totipotency—resides within the fertilized egg. But what drives the sequence of interlocking events that turns a single cell into a fully functioning adult? Decades of painstaking scientific investigation have taught us a great deal about the genetic and cell biological underpinnings of development. Unfortunately, we cannot begin to lay out the pieces of this biological puzzle here: we simply do not have the space to do it justice. However, one thing is very clear: the process of development is orchestrated by the genome contained within each fertilized egg. The sequence of its DNA directs the production of a variety of distinct cell types, each expressing different sets of genes and arranged in a precise, intricate, three-dimensional pattern. And the same genes that drive the establishment of this stunning array of cell types also endow these cells with the ability to assemble and work together to form the functionally distinct tissues of the body. Tissues Are Organized Mixtures of Many Cell Types Although the specialized tissues in our body differ in many ways, they all have certain basic requirements, usually fulfilled by a mixture of cell types, as illustrated for the skin in Figure 20–33. As discussed earlier, all tissues need mechanical strength, which is often supplied by a supporting framework of connective tissue laid down and inhabited by fibroblasts and related cell types. In this connective tissue, blood vessels lined with endothelial cells satisfy the need for oxygen, nutrients, and waste disposal. Likewise, most tissues are innervated by nerve cell axons, which are ensheathed by Schwann cells, some of which wrap around large axons to provide electrical insulation. Macrophages dispose of dead and damaged cells and other unwanted debris, and, together with lymphocytes and other white blood cells, they help combat infection. Most of these cell types originate outside the tissue and invade it, either early in the course of its development (endothelial cells, nerve cell axons, and Schwann cells) or continuously throughout life (macrophages and other cells derived from the blood). Figure 20–33 Mammalian skin is made of a mixture of cell types. Schematic diagrams show the cellular architecture of the main layers of thick skin. Skin can be viewed as a large organ composed of two main tissues: epithelial tissue (the epidermis) on the outside, and connective tissue on the inside. The outermost layer of the epidermis consists of flat, dead cells, whose intracellular organelles have disappeared (see Figure 20–36). The underlying connective tissue consists of the tough dermis (from which leather is made) and the deeper, fatty hypodermis. The dermis and hypodermis are richly supplied with blood vessels and nerves; some of the nerves extend into the epidermis, as shown. A similar supporting apparatus is required to maintain the principal specialized cells of many tissues: the contractile cells of muscle, the secretory cells of glands, or the blood-forming cells of bone marrow, for example. Almost every tissue is therefore an intricate mixture of many cell types that must remain different from one another while coexisting in the same environment. Moreover, in almost all adult tissues, cells are continually dying and being replaced; throughout this hurly-burly of cell replacement and tissue renewal, the organization and operation of the tissue must be preserved. Three main factors contribute to this stability. 1. Cell communication: Each type of specialized cell continually monitors its environment for signals from other cells and adjusts its behavior accordingly; the proliferation and even the survival of most vertebrate cells depends on such social signals (discussed in Chapters 16 and 18). This communication ensures that new cells are produced and survive only when and where they are required. 2. Selective cell adhesion: Because different cell types have different cadherins and other cell adhesion molecules in their plasma membrane, they tend to stick selectively, by homophilic binding, to other cells of the same type. They may also form selective attachments to certain other cell types and to specific extracellular matrix components. The selectivity of these cell adhesions keeps cells in their proper positions. 3. Cell memory: As discussed in Chapter 8, specialized patterns of gene expression, evoked by signals that acted during embryonic development, are afterward stably maintained, so that cells autonomously preserve their distinctive character and pass it on to their progeny. A fibroblast divides to produce more fibroblasts, and an endothelial cell divides to produce more endothelial cells. QUESTION 20–6 Why does ionizing radiation stop cell division? Different Tissues Are Renewed at Different Rates Human tissues vary enormously in their rate and pattern of cell turnover. At one extreme is the intestinal epithelium, in which cells are replaced every 3 to 6 days. At the other extreme is nervous tissue, in which most of the nerve cells last a lifetime without replacement. Between these extremes there is a spectrum of different speeds and styles of tissue renewal. Bone has a turnover time of about 10 years, and it involves renewal of the matrix as well as of cells (see Figure 20–8): old bone matrix is slowly eaten away by a set of cells called osteoclasts, akin to macrophages, while new matrix is deposited by another set of cells, osteoblasts, which are related to fibroblasts. New red blood cells are generated continually by blood-forming precursor cells in the bone marrow; they are released into the bloodstream, where they recirculate continually for about 120 days before being removed and destroyed by phagocytic cells in the liver and spleen. In the skin, dead cells in the outer layers of the epidermis are continually flaking off and being replaced from below, so that the epidermis is renewed with a turnover time of about 2 months. And so on. Our life depends on these renewal processes, as evidenced by our response to excessive exposure to radiation. In high enough doses, ionizing radiation blocks cell division and thus halts tissue renewal: within a few days, the lining of the intestine, for example, becomes denuded of cells, leading to the devastating diarrhea and water loss characteristic of acute radiation sickness. Clearly, there must be elaborate control mechanisms that keep cell production and cell loss in balance in the normal, healthy adult body. Cancers originate through violation of these controls, allowing rare mutant cells in the self-renewing tissues to survive and proliferate prodigiously. To understand cancer, therefore, it is important to understand the normal social controls on cell turnover that cancer perverts. Stem Cells and Proliferating Precursor Cells Generate a Continuous Supply of Terminally Differentiated Cells Most of the specialized, differentiated cells that need continual replacement are themselves unable to divide. This is true of red blood cells, the epidermal cells in the upper layers of the skin, and the absorptive and goblet cells of the gut epithelium. Such cells are referred to as terminally differentiated: they lie at the dead end of their developmental pathway. Figure 20–34 When a stem cell divides, each daughter can either remain a stem cell (self-renewal) or go on to become terminally differentiated. The terminally differentiated cells usually develop from proliferating precursor cells (also called transit-amplifying cells) that divide a limited number of times before they terminally differentiate. Stem-cell divisions can also produce two stem cells or two precursor cells, as long as the pool of stem cells is maintained. The cells that replace the terminally differentiated cells that are lost are generated from a stock of proliferating precursor cells, which themselves usually derive from a much smaller number of self renewing stem cells. Stem cells are not differentiated and can divide without limit (or at least for the lifetime of the animal). When one of these cells does divide, each daughter has a choice: either it can remain a stem cell, or it can embark on a course leading to terminal differentiation, usually via a series of precursor-cell divisions (Figure 20–34). The job of the stem cells and precursor cells, therefore, is not to carry out the specialized function of the differentiated cells, but rather to produce cells that will. Both stem cells and proliferating precursor cells are usually retained in their resident tissue along with their differentiated progeny. Stem cells are mostly present in small numbers and often have a nondescript appearance, making them difficult to spot; in some tissues, specific molecular markers can help identify them. Despite being undifferentiated, stem cells and precursor cells are nonetheless developmentally restricted: under normal conditions, they stably express sets of transcription regulators that ensure that their differentiated progeny will be of the appropriate cell types. The pattern of cell replacement varies from one stem-cell-based tissue to another. In the lining of the small intestine, for example, the absorptive and secretory cells are arranged as a single-layered, simple epithelium covering the surfaces of the fingerlike villi that project into the gut lumen. This epithelium is continuous with the epithelium lining the crypts, which descends into the underlying connective tissue (Figure 20–35A). The stem cells lie near the bottom of the crypts, where they give rise mostly to proliferating precursor cells, which move upward in the plane of the epithelial sheet. As they move upward, the precursor cells terminally differentiate into absorptive or secretory cells, which are shed into the gut lumen and die when they reach the tips of the villi (Figure 20–35B). Figure 20–35 Renewal occurs continuously in the epithelial lining of the adult mammalian intestine. (A) Micrograph of a section of part of the lining of the small intestine, showing the villi and crypts. Mucus-secreting goblet cells (stained purple) are interspersed among the absorptive brush-border cells in the epithelium covering the villi. Smaller numbers of two other secretory cell types—enteroendocrine cells (not visible here), which secrete gut hormones, and Paneth cells, which secrete antibacterial proteins—are also present and derive from the same stem cells. (B) Drawings showing the pattern of cell turnover and the proliferation of stem cells and precursor cells. The stem cells (red) give rise mainly to proliferating precursor cells (yellow), which slide continuously upward and terminally differentiate into secretory (purple) or absorptive (blue) cells, which are shed from the tip of the villus. The stem cells also give rise directly to terminally differentiated Paneth cells (gray), which move down to the bottom of the crypt. A contrasting example is the epidermis, a stratified epithelium. In the epidermis, proliferating stem cells and precursor cells are confined to the basal layer, adhering to the basal lamina. The differentiating cells travel outward from their site of origin in a direction perpendicular to the plane of the cell sheet; terminally differentiated cells and their corpses are eventually shed from the skin surface (Figure 20–36). Figure 20–36 The epidermis of the skin is a stratified epithelium renewed from stem cells in its basal layer. (A) The basal layer contains a mixture of stem cells and dividing precursor cells that are produced from the stem cells. On emerging from the basal layer, the precursor cells stop dividing and move outward, progressively differentiating as they go. Eventually, the cells undergo a special form of cell death: the nucleus and other organelles disintegrate, and the cell shrinks to the form of a flattened scale, packed with keratin filaments. These scales are ultimately shed from the skin surface. (B) Light micrograph of a cross section through the sole of a human foot. QUESTION 20–7 Why do you suppose epithelial cells lining the gut are lost and replaced (renewed) frequently, whereas most neurons last for the lifetime of the organism? Often, a single type of stem cell gives rise to several types of differentiated progeny: the stem cells of the intestine, for example, produce absorptive cells, goblet cells, and several other secretory cell types. The process of blood-cell formation, or hematopoiesis, provides an extreme example of this phenomenon. All of the different cell types in the blood—both the red blood cells that carry oxygen and the many types of white blood cells that fight infection (Figure 20–37)— ultimately derive from a shared hematopoietic stem cell found in the bone marrow (Figure 20–38). Figure 20–37 Blood contains many circulating cell types, all derived from a single type of stem cell. A sample of blood is smeared onto a glass cover slip, fixed (see Panel 1–1, p. 12), and stained with a dye that mainly stains the nucleus blue and cytoplasm red. Microscopic examination reveals numerous small erythrocytes (red blood cells), which lack a nucleus and DNA. The nucleated cells are different types of white blood cell: lymphocytes, eosinophils, basophils, neutrophils, and monocytes. Blood smears of this kind are routinely used as a clinical test in hospitals to look for increases or decreases in specific types of blood cells; for example, an increase in specific types of white blood cells could signal infection, inflammatory disorders, or leukemia. (Courtesy of Peter Takizawa.) Figure 20–38 A hematopoietic stem cell divides to generate more stem cells, as well as various types of precursor cells (not shown) that proliferate and differentiate into the mature blood cell types found in the circulation. Note that monocytes give rise to both macrophages, which are found in many tissues of the body, and osteoclasts, which eat away bone matrix. Megakaryocytes give rise to blood platelets by shedding cell fragments (Movie 20.5). A large number of extracellular signal molecules are known to act at various points in this cell lineage to help control the production of each cell type and to maintain appropriate numbers of precursor cells and stem cells. Specific Signals Maintain Stem-Cell Populations Every stem-cell system requires control mechanisms to ensure that new differentiated cells are generated in the appropriate places and in the right numbers. The controls depend on extracellular signals exchanged between the stem cells, their progeny, and other cell types. These signals, and the intracellular signaling pathways they activate, fall into a surprisingly small number of families, corresponding to half a-dozen basic signaling mechanisms, some of which are discussed in Chapter 16. These few mechanisms are used again and again—in different combinations—evoking different responses in different contexts in both the embryo and the adult. Almost all these signaling mechanisms contribute to the task of maintaining the complex organization of a stem-cell system such as that of the intestine. In this system, a class of signal molecules known as the Wnt proteins serves to promote the proliferation of the stem cells and precursor cells at the base of each intestinal crypt (Figure 20–39). Cells in the crypt produce, in addition, other signals that act at longer range to prevent activation of the Wnt pathway outside the crypts. The crypt cells also exchange yet other signals that control cell diversification, so that some precursor cells differentiate into secretory cells while others become absorptive cells. Figure 20–39 The Wnt signaling pathway maintains the proliferation of the stem cells and precursor cells in the intestinal crypt. The Wnt proteins are secreted by cells in and around the crypt base, especially by the Paneth cells—a subclass of terminally differentiated secretory cells that are generated from the gut stem cells. Newly formed Paneth cells, which move down to the crypt bottom instead of up to the tip of the villus, have a dual function: they secrete antimicrobial peptides to keep infection at bay, and at the same time they provide the signals to sustain the stem-cell population. Disorders of these signaling mechanisms disrupt the structure of the gut lining. In particular, as we see later, defects in the regulation of Wnt signaling underlie colorectal cancer—the most common form of human intestinal cancer. Stem Cells Can Be Used to Repair Lost or Damaged Tissues Because stem cells can proliferate indefinitely and produce progeny that differentiate, they provide for both continual renewal of normal tissue and repair of tissue lost through injury. For example, by transfusing a few hematopoietic stem cells into a mouse whose own blood stem cells have been destroyed by irradiation, it is possible to fully repopulate the animal with new blood cells and ultimately keep it from dying of anemia or infection. A similar approach is used in the treatment of human leukemia with irradiation (or cytotoxic drugs) followed by bone marrow transplantation. Although stem cells taken directly from adult tissues such as bone marrow have already proven their clinical value, another type of stem cell, first identified through experiments in mice, may have even greater potential—both for treating and understanding human disease. It is possible, through cell culture, to derive from early mouse embryos an extraordinary class of stem cells called embryonic stem cells, or cells. Under appropriate conditions, these cells can be kept proliferating indefinitely in culture and yet retain nearly unrestricted ES developmental potential, and are thus said to be pluripotent: if the cells from the culture dish are put back into an early embryo, they can give rise to all the tissues and cell types in the body, including the reproductive germ-line cells. Their descendants in the embryo are able to integrate perfectly into whatever site they come to occupy, adopting the character and behavior that normal cells would show at that site. Such an approach can be used to study gene function: in this case, the ES cells are genetically manipulated—to inactivate a gene or insert a modified one—prior to being returned to an embryo (see Figure 10–30). ES cells can also be induced, by the appropriate extracellular signal molecules, to differentiate in culture into a large variety of cell types (Figure 20–40). Figure 20–40 Mouse ES cells can be induced to differentiate into specific cell types in culture. ES cells are harvested from the inner cell mass of an early mouse embryo and can be maintained indefinitely as pluripotent stem cells in culture. If they are allowed to aggregate (not shown) and are then exposed to the appropriate extracellular signal molecules, in the correct sequence and at the right time, these cells can be induced to differentiate into specific cell types of interest (Movie 20.6). Cells with properties similar to those of mouse ES cells can also be derived from early human embryos, and these cells can be induced to differentiate into a variety of cell types as illustrated in Figure 20–40. In principle, human ES cells provide a potentially inexhaustible supply of cells that might be used for the replacement or repair of mature human tissues that are damaged. There are, however, many hurdles to be cleared before such dreams can become reality. One major problem concerns immune rejection: if the transplanted cells are genetically different from the cells of the person into whom they are grafted, they are likely to be rejected and destroyed by the immune system. Beyond the practical scientific difficulties, there are also ethical concerns about the use of human embryos to produce human ES cells. One way around both of these problems is to generate human pluripotent cells in a different way, as we now discuss. Induced Pluripotent Stem Cells Provide a Convenient Source of Human ES-like Cells It is now possible to produce pluripotent stem cells without the use of embryos. Differentiated cells can be taken from an adult mouse or human tissue, grown in culture, and reprogrammed into an ES-like state by artificially driving the expression of a set of three transcription regulators: Oct4, Sox2, and Klf4 (see Figure 8–21). This treatment is sufficient to permanently convert fibroblasts into cells with practically all the properties of ES cells, including the ability to proliferate indefinitely, differentiate in diverse ways, and—in the case of mouse cells—contribute to the formation of any tissue. These ES-like cells are called induced pluripotent stem (iPS) cells. Techniques for efficiently producing and precisely directing the differentiation of human iPS cells are continually being refined with an eye toward developing effective cell-based therapies. Experiments in mice suggest that it should be possible to use cells derived from human iPS cells to replace the skeletal muscle fibers that degenerate in individuals with muscular dystrophy, the nerve cells that die in people with Parkinson’s disease, the insulin-secreting cells that are destroyed by the immune system in type 1 diabetics, and the cardiac muscle cells that are lost during a heart attack. Perhaps one day it might even become possible to grow entire organs from human iPS cells by a recapitulation of embryonic development, as we discuss shortly. In the meantime, human iPS cells are proving invaluable in other ways. They can be used to generate large, homogeneous populations of differentiated human cells of specific types in culture; these can be used to test for potential toxic or beneficial effects of candidate drugs. It is even possible to generate iPS cells from people who suffer from a particular genetic disease and to use these iPS cells to produce affected, differentiated cell types, which can then be studied to learn more about the disease mechanism and to search for potential treatments (Figure 20–41). Figure 20–41 iPS cells can be used to study and treat genetic disease. Genes encoding a combination of transcription regulators can be used to reprogram skin fibroblasts harvested from an individual with a genetic disorder, causing them to form iPS cells that contain the disease-causing mutation. The patient-specific iPS cells can then be induced to differentiate into the type of cell affected by the disorder, enabling studies of the disease mechanism and the search for potential treatments (as shown on the left side of the diagram). Patient-derived iPS cells could also someday themselves form the basis of disease treatment (as shown on the right side of the diagram). In this case, repair of the disease-causing mutation could lead to the production of healthy cells that would then be transplanted into the individual without fear of immune rejection, as the cells were initially derived from that individual. (Adapted from D.A. Robinton and G.Q. Daley, Nature 481:295–305, 2012.) Such an approach has led to insights into Timothy syndrome, a rare genetic disease caused by mutations in a gene that encodes a specific type of Ca2+ channel. The altered channel fails to close properly after opening, leading to multiple defects, including abnormal heart rhythm and, in some individuals, autism. The iPS cells produced from such individuals have been coaxed to differentiate in culture into neurons and heart muscle cells, which are now being used to study the physiological consequences of the Ca2+ channel abnormality and to hunt for drugs that can correct the defects. In addition, studies of the gene expression patterns of ES and iPS cells themselves are illuminating some of the many unsolved mysteries of developmental and stem-cell biology, including the molecular mechanisms that maintain pluripotency or that restrict specific developmental fates. Mouse and Human Pluripotent Stem Cells Can Form Organoids in Culture Under the appropriate conditions, ES cells and iPS cells from mice and humans can be made to proliferate, differentiate, and self assemble to form small, three-dimensional structures called organoids. These miniature organs, grown in a culture dish, resemble the normal organs to a remarkable degree in terms of their organization. In one striking example, shown in Figure 20–42, human ES cells form an eyelike structure that includes a multilayered retina similar in organization to that seen in the developing human eye. Figure 20–42 Cultured ES cells can give rise to a three dimensional organoid. (A) Schematic drawing shows how, under appropriate conditions, mouse or human pluripotent cells in culture can proliferate, differentiate, and self-assemble to form a three-dimensional, eye-like structure (an optic cup), which includes a multilayered retina similar in organization to the one that forms during normal eye development in vivo. (B) Fluorescence micrograph of an optic cup formed by human ES cells in culture. The structure includes a developing retina containing multiple layers of neural cells (stained green) and an underlying layer of pigmented epithelium, the apical surface of which is stained red. All nuclei are stained blue. (A, adapted from M. Eiraku and Y. Sasai, Curr. Opin. Neurobiol. 22:768 777, 2012; B, from T. Nakano et al., Cell Stem Cell 10:771–785 2012.) Mouse and human iPS cells, and precursor cells derived from them, have now been used to form organoids that resemble a variety of developing organs, including the human brain, arguably the most complex and sophisticated structure on Earth. Such organoids provide powerful models for studying organ development in a culture dish, where one can identify and manipulate the genes involved and explore the roles of cell–cell interactions in ways not possible in an intact organism. In addition, organoids can be used to investigate how developmental pathways can be derailed by disease. For example, brain organoids have been produced using human iPS cells derived from an individual with microcephaly, a condition characterized by severely stunted brain growth and development. Careful analysis of these developing organoids revealed that the microcephaly in this case was probably caused by the premature cessation of proliferation and differentiation of precursor cells, resulting in a decreased production of brain cells. The development of iPS cells and organoid technology has opened up an entirely new way to study human development and disease. It also opens up promising avenues for treatment. Glossary differentiated cell Cell that has undergone a coordinated change in gene expression, enabling it to perform a specialized function. stem cell Relatively undifferentiated, self-renewing cell that produces daughter cells that can either differentiate into more specialized cell types or can retain the developmental potential of the parent cell. Wnt protein Member of a family of extracellular signal molecules that regulates cell proliferation and migration during embryonic development and that helps to maintain stem cells in a proliferative state. pluripotent Capable of giving rise to any type of cell or tissue. induced pluripotent stem (iPS) cell Somatic cell that has been reprogrammed to resemble and behave like a pluripotent embryonic stem (ES) cell through the artificial introduction of a set of genes encoding particular transcription regulators. organoid A miniature, three-dimensional collection of tissues formed from the proliferation, differentiation, and self-assembly of pluripotent cells in culture. embryonic stem (ES) cell An undifferentiated cell type derived from the inner cell mass of an early mammalian embryo and capable of differentiating to give rise to any of the specialized cell types in the adult body. embryonic stem (ES) cell An undifferentiated cell type derived from the inner cell mass of an early mammalian embryo and capable of differentiating to give rise to any of the specialized cell types in the adult body