Chapter 22:  Histology: The Lives and Deaths of Cells in Tissues

Epidermal Cells Form a Multilayered Waterproof Barrier

The epidermis suffers more direct, frequent, and damaging encounters with the external world than any other tissue in the body. Its need for repair and renewal is central to its organization.

The epidermis is a multilayered (stratified) epithelium composed largely of keratinocytes (so named because their characteristic differentiated activity is the synthesis of intermediate filament proteins called keratins, which give the epidermis its toughness) (Figure 22-2). These cells change their appearance from one layer to the next. Those in the innermost layer, attached to an underlying basal lamina, are termed basal cells, and it is usually only these that divide. Above the basal cells are several layers of larger prickle cells (Figure 22-3), whose numerous desmosomes—each a site of anchorage for thick tufts of keratin filaments—are just visible in the light microscope as tiny prickles around the cell surface (hence the name). Beyond the prickle cells lies the thin, darkly staining granular cell layer (see Figure 22-2). It is at this level that the cells are sealed together to form a waterproof barrier, fulfilling the most fundamentally important of all the functions of the epidermis. Mice that fail to form this barrier because of a genetic defect die from rapid fluid loss soon after birth, even though their skin appears normal in other respects.

The granular layer, with its barrier to the movement of water and solutes, marks the boundary between the inner, metabolically active strata and the outermost layer of the epidermis, consisting of dead cells whose intracellular organelles have disappeared. These outermost cells are reduced to flattened scales, or squames, filled with densely packed keratin. The plasma membranes of both the squames and the outer granular cells are reinforced on their cytoplasmic surface by a thin (12 nm), tough, cross-linked layer of proteins, including a cytoplasmic protein called involucrin. The squames themselves are normally so compressed and thin that their boundaries are hard to make out in the light microscope, but soaking in sodium hydroxide solution (or a warm bath tub) makes them swell slightly, and their outlines can then be seen (see Figure 22-2).

Differentiating Epidermal Cells Synthesize a Sequence of Different Keratins as They Mature

Having described the static picture, let us now set it in motion and see how the epidermis is continually renewed by the production of new cells in the basal layer. While some basal cells are dividing, adding to the population in the basal layer, others (their sisters or cousins) are slipping out of the basal cell layer into the prickle cell layer, taking the first step on their outward journey. When they reach the granular layer, the cells start to lose their nucleus and cytoplasmic organelles, through a degradative mechanism that involves partial activation of the machinery of apoptosis; in this way, the cells are transformed into the keratinized squames of the keratinized layer. These finally flake off from the surface of the skin (and become a main constituent of household dust). The period from the time a cell is born in the basal layer of the human skin to the time it is shed from the surface is of the order of a month, depending on the region of the body.

The accompanying molecular transformations can be studied by analyzing either thin slices of epidermis cut parallel to the surface or successive layers of cells stripped off by repeatedly applying and removing strips of adhesive tape. The keratin molecules, for example, which are plentiful in all layers of the epidermis, can be extracted and characterized. They are of many types (discussed in Chapter 16), encoded by a large family of homologous genes, with the variety further increased through alternative RNA splicing. As the new keratinocyte in the basal layer is transformed into the squame in the outermost layers (see Figure 22-3), it switches from one selection of keratins to another. Meanwhile other characteristic proteins, such as involucrin, also begin to be synthesized as part of a coordinated program of terminal cell differentiation—the process in which a precursor cell acquires its final specialized characteristics and usually permanently stops dividing. The whole program is initiated in the basal layer. It is here that the fates of the cells are decided.

Epidermis Is Renewed by Stem Cells Lying in Its Basal Layer

The outer layers of the epidermis are replaced a thousand times over in the course of a human lifetime. In the basal layer there have to be cells that can remain undifferentiated and carry on dividing for this whole period, continually throwing off descendants that differentiate, leave the basal layer, and are eventually discarded. The process can be maintained only if the basal cell population is self-renewing. It must therefore contain some cells that generate a mixture of progeny, including daughters that remain undifferentiated like their parent, as well as daughters that differentiate. Cells with this property are called stem cells. They have so important a role in such a variety of tissues that it is useful to have a formal definition.

The defining properties of a stem cell are as follows:

• 1

  It is not itself terminally differentiated (that is, it is not at the end of a pathway of differentiation).

• 2

  It can divide without limit (or at least for the lifetime of the animal).

• 3
  When it divides, each daughter has a choice: it can either remain a stem cell, or it can embark on a course that commits it to terminal differentiation (Figure 22-4).

Although it is part of the definition of a stem cell that it should be able to divide, it is not part of the definition that it should divide rapidly; in fact, stem cells usually divide at a relatively low rate. They are required wherever there is a recurring need to replace differentiated cells that cannot themselves divide, and this includes a great variety of tissues. Thus stem cells are of many types, specialized for the genesis of different classes of terminally differentiated cells—epidermal stem cells for epidermis, intestinal stem cells for intestinal epithelium, hemopoietic stem cells for blood, and so on. Each stem-cell system nevertheless raises similar fundamental questions. What factors determine whether the stem cell divides or stays quiescent? What decides whether a given daughter cell differentiates or remains a stem cell? And where the stem cell can give rise to more than one kind of differentiated cell—as is very often the case—what determines which differentiation pathway is followed?

The Two Daughters of a Stem Cell Do Not Always Have to Become Different

At steady state, to maintain a stable stem-cell population, precisely 50% of the daughters of stem cells in each cell generation must remain as stem cells. In principle, this could be achieved in two ways—through environmental asymmetry or through divisional asymmetry (Figure 22-5). In the one strategy, the division of a stem cell could generate two initially similar daughters whose fates would be governed by their subsequent environment; 50% of the population of daughters would remain as stem cells, but the two daughters of an individual stem cell in the population might often have the same fate. At the opposite extreme, the stem cell division could be always strictly asymmetric, producing one daughter that inherits the stem-cell character and another that inherits factors that force it to embark on differentiation. In the latter case, the existing stem cells could never increase their numbers, and any loss of stem cells would be irreparable.

In fact, if a patch of epidermis is destroyed, the damage is repaired by surrounding epidermal cells that migrate in and proliferate to cover the denuded area. In this process, a new self-renewing patch of epidermis is established, implying that additional stem cells have been generated to make up for the loss. These must have been produced by symmetric divisions in which one stem cell gives rise to two. In this way, the stem cell population adjusts its numbers to fit the available niche.

Observations such as these suggest that the maintenance of stem cell character in the epidermis might be controlled by contact with the basal lamina, with a loss of contact triggering the start of terminal differentiation, and maintenance of contact tending to preserve stem cell potential. This idea contains a grain of truth, but it is not the whole truth, as we now explain.

The Basal Layer Contains Both Stem Cells and Transit Amplifying Cells

Basal keratinocytes can be dissociated from intact epidermis and can proliferate in a culture dish, giving rise to new basal cells and to terminally differentiated cells. Even within a population of cultured basal keratinocytes that all seem undifferentiated, there is great variation in the ability to proliferate. When cells are taken singly and tested for their ability to found new colonies, some seem unable to divide at all, others go through only a few division cycles and then halt, and still others divide enough times to form large colonies. This proliferative potential directly correlates with the expression of the β1 subunit of integrin (see Figure 19-64). Clusters of cells with high levels of this molecule can be found in the basal layer of the intact human epidermis also, and they are thought to be the stem cells (Figure 22-6).

Basal cells expressing β1 integrin at a lower level can also divide—indeed, they divide more frequently—but only for a limited number of division cycles, after which they leave the basal layer and differentiate. These latter cells are called transit amplifying cells—“transit”, because they are in transit from a stem-cell character to a differentiated character; “amplifying”, because the division cycles they go through have the effect of amplifying the number of differentiated progeny that result from a single stem-cell division (Figure 22-7). Mingled with the population of transit amplifying cells are some cells, still connected with the basal lamina by a thin stalk, that have already stopped dividing and begun to differentiate, as indicated by the types of keratin molecules they express. Contact with the basal lamina, therefore, cannot be the only factor controlling the developmental fate of an epidermal basal cell.

This is not to say that contact with the basal lamina or a similar substratum does not matter. If cultured basal keratinocytes are held in suspension, instead of being allowed to settle and attach to the bottom of the culture dish, they all stop dividing and differentiate. To remain as an epidermal stem cell, it is apparently necessary for it to be attached to the basal lamina or other extracellular matrix, even though it is not sufficient. This requirement helps ensure that the size of the stem cell population does not increase without limit. If crowded out of their regular niche on the basal lamina, the cells lose their stem cell character. When this rule is broken, as in some cancers, the result can be an ever-growing tumor.

Epidermal Renewal Is Governed by Many Interacting Signals

Cell turnover in the epidermis seems at first glance a simple matter, but the simplicity, as we have just seen, is deceptive. There are many points in the process that have to be controlled according to circumstances: the rate of stem-cell division; the probability that a stem-cell daughter will remain a stem cell; the number of cell divisions of the transit amplifying cells; the timing of exit from the basal layer, and the time that the cell then takes to complete its differentiation program and be sloughed from the surface. Regulation of these steps must enable the epidermis to respond to rough usage by becoming thick and callused, and to repair itself when wounded. In specialized regions of epidermis, such as those that form hair follicles, with their own specialized subtypes of stem cells, yet more controls are needed to organize the local pattern.

Each control point is important in its own way, and a whole panoply of molecular signals regulate them, so as to keep the body surface always properly covered. Most of the cell communication mechanisms described in Chapter 15 are implicated, either in signaling between cells within the epidermis or in signaling between epidermis and dermis. The EGF, FGF, Wnt, Hedgehog, Notch, BMP/TGFβ, and integrin signaling pathways are all involved (and we shall see that the same is true of most other tissues). Mutations in components of the Hedgehog or Wnt pathways, for example, can lead to the development of epidermal cancers. Components of the Hedgehog, Notch, BMP, and Wnt pathways when misexpressed interfere with the formation of hairs, blocking their development or causing them to develop out of place.

Activation of the Wnt pathway seems to favor maintenance of stem-cell character, inhibiting the switch from stem cell to transit amplifying cell, whereas Notch signaling in the epidermis seems to have a contrary effect, inhibiting neighbors of stem cells from remaining as stem cells. TGFβ has a key role in signaling to the dermis during the repair of skin wounds, promoting the formation of collagen-rich scar tissue. And integrins in the epidermis are not merely markers of cell character, but also regulators of cell fate. Thus, when transgenic mice are engineered to maintain in upper epidermal layers the expression of integrins normally confined to the basal layer, they develop a condition resembling the common human skin disorder psoriasis: the rate of basal cell proliferation is greatly increased, the epidermis thickens, and cells are shed from the surface of the skin within as little as a week after emerging from the basal layer, before they have had time to keratinize fully. The precise individual functions of all the various signaling mechanisms in the epidermis are only beginning to be disentangled.

The Mammary Gland Undergoes Cycles of Development and Regression

In specialized regions of the body surface, other types of cells besides the keratinized cells described above develop from the embryonic epidermis. In particular, secretions such as sweat, tears, saliva, and milk are produced by cells segregated in deep-lying glands that originate as ingrowths of the epidermis. These epithelial structures have functions and patterns of renewal quite different from those of keratinizing regions.

The mammary glands are the largest and most remarkable of these secretory organs. They are the defining feature of mammals and an important concern in many ways: not only for nourishment of babies and attraction of the opposite sex, but also as the basis for a large industry—the dairy industry—and as the site of some of the commonest forms of cancer. Mammary tissue illustrates most dramatically that developmental processes continue in the adult body; and it shows how cell death by apoptosis can allow development to go into reverse.

Milk production must be switched on when a baby is born and switched off when the baby is weaned. A “resting” adult mammary gland consists of branching systems of ducts embedded in fatty connective tissue. The ducts are lined by an epithelium that includes a subpopulation of mammary stem cells (though the precise location of the stem cells is still debated). As a first step toward milk production, the hormones that circulate during pregnancy cause the duct cells to proliferate, increasing their numbers tenfold or twentyfold. The terminal portions of the ducts grow and branch, forming little dilated outpocketings, or alveoli, containing secretory cells (Figure 22-8). Milk secretion begins only when these cells are stimulated by the different combination of hormones that circulate in the mother after the birth of the baby. A further tier of hormonal control governs the actual ejection of milk from the breast: the stimulus of suckling causes cells of the hypothalamus (in the brain) to release the hormone oxytocin, which travels via the bloodstream to act on myoepithelial cells. These musclelike cells originate from the same epithelial precursor population as the secretory cells of the breast, and they have long spidery processes that embrace the alveoli. In response to oxytocin they contract, thereby squirting milk out of the alveoli into the ducts.

Eventually, when the baby is weaned and suckling stops, the secretory cells die by apoptosis, and most of the alveoli disappear. Macrophages rapidly clear away the dead cells, and the gland reverts to its resting state. This ending of lactation is abrupt and, unlike the events that lead up to it, seems to be induced by the accumulation of milk, rather than by a hormonal mechanism. If one subset of mammary ducts is obstructed so that no milk can be discharged, the secretory cells that supply it commit mass suicide by apoptosis, while other regions of the gland survive and continue to function. The apoptosis is triggered by a combination of factors including TGFβ3, which accumulates where milk secretion is blocked (Figure 22-9).

Cell division in the growing mammary gland is regulated not only by hormones but also by local signals passing between cells within the epithelium and between the epithelial cells and the connective tissue, or stroma, in which the epithelial cells are embedded. All the signals listed earlier as important in controlling cell turnover in the epidermis are also implicated in controlling events in the mammary gland. Again, signals delivered via integrins play a crucial part: deprived of the basal lamina adhesions that activate integrin signaling, the epithelial cells fail to respond normally to hormonal signals. Faults in these interacting control systems underlie some of the commonest forms of cancer, and we need to understand them better.

Summary

Skin consists of a tough connective tissue, the dermis, overlaid by a multilayered waterproof epithelium, the epidermis. The epidermis is continually renewed from stem cells, with a turnover time, in humans, of the order of a month. Stem cells, by definition, are not terminally differentiated and have the ability to divide throughout the lifetime of the organism, yielding some progeny that differentiate and others that remain stem cells. The epidermal stem cells lie in the basal layer, attached to the basal lamina. Progeny that become committed to differentiation go through several rapid divisions in the basal layer, and then stop dividing and move out toward the surface of the skin. They progressively differentiate, switching from expression of one set of keratins to expression of another until, eventually, their nuclei degenerate, producing an outer layer of dead keratinized cells that are continually shed from the surface.

The fate of the daughters of a stem cell is controlled in part by interactions with the basal lamina and in part by a variety of signals from neighboring cells. These controls allow two stem cells to be generated from one during repair processes, and they regulate the rate of basal cell proliferation according to need. Glands connected to the epidermis, such as the mammary glands, have their own stem cells and their own distinct patterns of cell turnover, governed by other conditions. In the breast, for example, circulating hormones stimulate the cells to proliferate, differentiate, and make milk; the cessation of suckling triggers the milk-secreting cells to die by apoptosis, as a result of a build-up of TGFβ3 where milk fails to be drained away.

Olfactory Sensory Neurons Are Continually Replaced

In the olfactory epithelium of the nose (Figure 22-10A), a subset of the epithelial cells differentiate as olfactory sensory neurons. These cells have modified, immotile cilia on their free surfaces (see Figure 15-43), containing odorant receptor proteins, and a single axon extending from their basal end towards the brain (Figure 22-10B). The neurons are held in place and separated from one another by supporting cells that span the thickened epithelium and have properties similar to those of glial cells in the central nervous system. The sensory surfaces are kept moist and protected by a layer of fluid secreted by cells sequestered in glands that communicate with the exposed surface. Even with this protection, however, each olfactory neuron survives for only a month or two, and so a third class of cells—the basal cells—is present in the epithelium to generate replacements for the olfactory neurons that are lost. The basal cells are stem cells for the production of the neurons.

As discussed in Chapter 15, each olfactory neuron expresses most probably only one of the hundreds of odorant receptor genes in the genome. This determines which odorants the cell responds to. But regardless of the odor, the response of every olfactory neuron is the same—a train of action potentials sent back along its axon to the brain. The discriminating sensibility of an individual olfactory neuron is therefore useful only if its axon delivers its messages to the specific relay station in the brain that is dedicated to the particular range of odors that the neuron senses. These relay stations are called glomeruli. They are located in structures called the olfactory bulbs (one on each side of the brain), with about 1800 glomeruli in each bulb (in the mouse). Olfactory neurons expressing the same odorant receptor are widely scattered in the olfactory epithelium, but their axons all converge on the same glomerulus (Figure 22-10C). As new olfactory neurons are generated, replacing those that die, they must in turn send their axons to the correct glomerulus. The odorant receptor proteins thus have been proposed to have a second function: helping to guide the growing tips of the new axons along specific paths to the appropriate target glomeruli in the olfactory bulbs. If it were not for the continual operation of this guidance system, a rose might smell in one month like a lemon, in the next like rotting fish.

Auditory Hair Cells Have to Last a Lifetime

The sensory epithelium responsible for hearing is the most precisely and minutely engineered of all the tissues in the body (Figure 22-11). Its sensory cells, the auditory hair cells, are held in a rigid framework of supporting cells and overlaid by a mass of extracellular matrix (the tectorial membrane), in a structure called the organ of Corti. The hair cells convert mechanical stimuli into electrical signals. Each has a characteristic organ-pipe array of giant microvilli (called stereocilia) protruding from its surface as rigid rods, filled with cross-linked actin filaments, and arranged in ranks of graded height. The dimensions of each such array are specified with extraordinary accuracy according to the location of the hair cell in the ear and the frequency of sound that it has to respond to. Sound vibrations rock the organ of Corti, causing the bundles of stereocilia to tilt (Figure 22-12) and mechanically gated ion channels in the membranes of the stereocilia to open or close (Figure 22-13). The flow of electric charge carried into the cell by the ions alters the membrane potential and thereby controls the release of neurotransmitter at the cell's basal end, where the cell synapses with a nerve ending.

In humans and other mammals, the auditory hair cells, unlike olfactory neurons, have to last a lifetime. If they are destroyed by disease, toxins, or excessively loud noise, they are not regenerated and the resultant hearing loss is permanent. But in other vertebrates, when auditory hair cells are destroyed, the supporting cells are triggered to divide and behave as stem cells, generating progeny that can differentiate as replacements for the hair cells that are lost. With better understanding of how this regeneration process is regulated, we may one day be able to induce the auditory epithelium to repair itself in humans also.

Most Permanent Cells Renew Their Parts: the Photoreceptor Cells of the Retina

The neural retina is the most complex of the sensory epithelia. It consists of several cell layers organized in a way that seems perverse. The neurons that transmit signals from the eye to the brain (called retinal ganglion cells) lie closest to the external world, so that the light, focused by the lens, must pass through them to reach the photoreceptor cells. The photoreceptors, which are classified as rods or cones, according to their shape, lie with their photoreceptive ends, or outer segments, partly buried in the pigment epithelium (Figure 22-14). Rods and cones contain different rhodopsins—photosensitive complexes of opsin protein with the visual pigment retinal. Rods are especially sensitive at low light levels, while cones (of which there are three types, each with a different opsin, giving a different spectral response) detect color and fine detail.

The outer segment of a photoreceptor appears to be a modified cilium with a characteristic ciliumlike arrangement of microtubules in the region where the outer segment is connected to the rest of the cell (Figure 22-15). The remainder of the outer segment is almost entirely filled with a dense stack of membranes in which the photosensitive complexes are embedded; light absorbed here produces an electrical response, as discussed in Chapter 15. At their opposite ends, the photoreceptors form synapses on interneurons, which relay the signal to the retinal ganglion cells (see Figure 22-14).

Photoreceptors in humans, like human auditory hair cells, are permanent cells that do not divide and are not replaced if destroyed by disease or by a mis-directed laser beam. But the photosensitive rhodopsin molecules are not permanent but are continually degraded and replaced. In rods (although not, curiously, in cones), this turnover is organized in an orderly production line, which can be analyzed by following the passage of a cohort of radiolabeled protein molecules through the cell after a short pulse of radioactive amino acid (Figure 22-16). The radiolabeled proteins can be followed from the Golgi apparatus in the inner segment of the cell to the base of the stack of membranes in the outer segment. From here they are gradually displaced toward the tip as new material is fed into the base of the stack. Finally (after about 10 days in the rat), on reaching the tip of the outer segment, the labeled proteins and the layers of membrane in which they are embedded are phagocytosed (chewed off and digested) by the cells of the pigment epithelium.

This example illustrates a general point: even though individual cells of certain types persist, very little of the adult body consists of the same molecules that were laid down in the embryo.

Summary

Most sensory receptor cells, like epidermal cells and nerve cells, derive from the epithelium forming the outer surface of the embryo. They transduce external stimuli into electrical signals, which they relay to neurons via chemical synapses. Olfactory receptor cells in the nose are themselves full-fledged neurons, sending their axons to the brain. They have a lifetime of only a month or two, and are continually replaced by new cells derived from stem cells in the olfactory epithelium. Each olfactory neuron expresses just one of the hundreds of different olfactory receptor proteins for which genes exist in the genome, and the axons from all olfactory neurons expressing the same receptor protein navigate to the same glomeruli in the olfactory bulbs of the brain.

Auditory hair cells—the receptor cells for sound—unlike olfactory receptor cells, have to last a lifetime, in mammals at least. They have no axon but make synaptic contact with nerve terminals in the auditory epithelium. They take their name from the hair-like bundle of stereocilia (giant microvilli) on their outer surface. Sound vibrations tilt the bundle, pulling mechanically gated ion channels on the stereocilia into an open configuration to excite the cell electrically.

Photoreceptor cells in the retina of the eye absorb photons in rhodopsin molecules held in stacks of membrane in the photoreceptor outer segments, triggering an electrical excitation by a more indirect intracellular signaling pathway. Although the photoreceptor cells themselves are permanent and irreplaceable, the stacks of rhodopsin-rich membrane that they contain undergo continual renewal.

Adjacent Cell Types Collaborate in the Alveoli of the Lungs

The airways of the lungs are formed by repeated branching of a system of tubes that originated in the embryo from an outpocketing of the gut lining, as discussed in Chapter 21. Repeated tiers of branching terminate in several hundred million air-filled sacs—the alveoli. Alveoli have thin walls, closely apposed to the walls of blood capillaries so as to allow exchange of O₂ and CO₂ with the blood stream (Figure 22-17).

To survive, the cells lining the alveoli must remain moist. At the same time, they must serve as a gas container that can expand and contract with each breath in and out. This creates a problem. When two wet surfaces touch, they become stuck together by surface tension in the layer of water between them—an effect that operates more powerfully the smaller the scale of the structure. There is a risk, therefore, that the alveoli may collapse and be impossible to reinflate. The problem is solved by the presence of two types of cells in the lining of the alveoli. Type I alveolar cells cover most of the wall: they are thin and flat (squamous) to allow gas exchange. Type II alveolar cells are interspersed among them. These are plump and secrete surfactant, a phospholipid-rich material that forms a film on the free water surfaces and reduces surface tension, making the alveoli easy to reinflate even if they collapse. The production of adequate amounts of surfactant in the fetus, starting at about 5 months of pregnancy in humans, marks the beginning of the possibility of independent life. Premature babies born before this stage are unable to inflate their lungs and breathe; those born after it can do so and, with intensive care, can survive.

Goblet Cells, Ciliated Cells, and Macrophages Collaborate to Keep the Airways Clean

Higher up in the airways one finds different combinations of cell types, serving different purposes. The air we breathe is full of dust, dirt, and air-borne microorganisms. To keep the lungs clear and healthy, this debris must be constantly swept out. To perform this task, the larger airways are lined by a relatively thick respiratory epithelium (Figure 22-18). This includes three differentiated cell types: goblet cells (so named because of their shape), which secrete mucus, ciliated cells, with cilia that beat, and a small number of endocrine cells, secreting serotonin and peptides that act as local mediators. These signal molecules affect nerve endings and other neighboring cells in the respiratory tract, so as to help regulate the rate of mucus secretion and ciliary beating, the contraction of surrounding smooth muscle cells that can constrict the airways, and other functions. Basal cells are also present, and serve as stem cells for renewal of the epithelium.

The mucus secreted by the goblet cells forms a viscoelastic blanket about 5 μm thick over the tops of the cilia. The cilia, all beating in the same direction, at a rate of about 12 beats per second, sweep the mucus out of the lungs, carrying with it the debris that has become stuck to it. This conveyor belt for the removal of rubbish from the lungs is called the mucociliary escalator. Of course, some inhaled particles may reach the alveoli themselves, where there is no escalator. Here, the unwanted matter is removed by yet another class of specialized cells, macrophages, which roam the lungs and engulf foreign matter and kill and digest bacteria. Many millions of macrophages, loaded with debris, are swept out of the lungs every hour on the mucociliary escalator.

At the upper end of the respiratory tract, the wet mucus-covered respiratory epithelium gives way abruptly to stratified squamous epithelium. This cell sheet is structured for mechanical strength and protection, and, like epidermis, it consists of many layers of flattened cells densely packed with keratin. It differs from epidermis in that it is kept moist and its cells retain their nucleus even in the outermost layers. Abrupt boundaries of epithelial cell specialization, such as that between the mucous and the stratified squamous epithelium of the respiratory tract, are also found in other parts of the body, but very little is known about how they are created and maintained.

The Lining of the Small Intestine Renews Itself Faster Than Any Other Tissue

Only air-breathing vertebrates have lungs, but all vertebrates, and almost all invertebrate animals, have a gut—that is, a digestive tract lined with cells specialized for the digestion of food and absorption of the nutrient molecules released by the digestion. These two activities are hard to carry on at the same time, as the processes that digest food in the lumen of the gut are liable also to digest the lining of the gut itself, including the cells that absorb the nutrients. The gut uses several strategies to solve the problem.

The fiercest digestive processes, involving acid hydrolysis as well as enzyme action, are conducted in a separate reaction vessel, the stomach. The products are then passed on to the small intestine, where the nutrients are absorbed and enzymatic digestion continues, but at a neutral pH. The different regions of the gut lining consist of correspondingly different mixtures of cell types. The stomach epithelium includes cells that secrete acid, and other cells that secrete digestive enzymes that work at acid pH. Conversely, glands (in particular the pancreas) that discharge into the initial segment of the small intestine contain cells that secrete bicarbonate to neutralize the acidity, along with other cells that secrete digestive enzymes that work at neutral pH. The lining of the intestine, downstream from the stomach, contains both absorptive cells and cells specialized for secretion of mucus, which covers the epithelium with a protective coat. In the stomach, too, the most exposed surfaces are lined with mucous cells. And, in case these measures are not enough, the whole lining of the stomach and intestine is continually renewed and replaced by freshly generated cells, with a turnover time of a week or less.

The renewal process has been studied best in the small intestine (Figure 22-19). The lining of the small intestine (and of most other regions of the gut) is a single-layered epithelium. This epithelium covers the surfaces of the villi that project into the lumen, and it lines the crypts that descend into the underlying connective tissue. Dividing stem cells lie in a protected position in the depths of the crypts. These generate four types of differentiated progeny (Figure 22-20): (1) absorptive cells (also called brush-border cells), with densely packed microvilli on their exposed surfaces to increase their active surface area; (2) goblet cells (as in respiratory epithelium), secreting mucus; (3) Paneth cells, forming part of the innate immune defense system (discussed in Chapter 25) and secreting (along with some growth factors) cryptdins—proteins of the defensin family that kill bacteria (see Figure 25-39); and (4) enteroendocrine cells, of more than 15 different subtypes, secreting serotonin and peptide hormones, such as cholecystokinin, that act on neurons and other cell types in the gut wall and regulate the growth, proliferation and digestive activities of cells of the gut and other tissues; many of these gut hormones function also as neuropeptide signal molecules in the nervous system.

The absorptive, goblet, and enteroendocrine cells travel mainly upward from the stem-cell region, by a sliding movement in the plane of the epithelial sheet, to cover the surfaces of the villi. As in the epidermis, there is a transit amplifying stage of cell proliferation: on their way out of the crypt, the precursor cells, already committed to differentiation, go through four to six rapid divisions before they stop dividing and differentiate terminally. Within 2–5 days (in the mouse) after emerging from the crypts, the cells reach the tips of the villi, where they undergo the initial stages of apoptosis and are finally discarded into the gut lumen. The Paneth cells are produced in much smaller numbers and have a different migration pattern. They stay down at the bottom of the crypts, where they too are continually replaced, although not so rapidly, persisting for about 20 days (in the mouse) before undergoing apoptosis and being phagocytosed by their neighbors.

The driving force for the movements of the gut epithelial cells is still a mystery. Their different patterns of migration may be controlled by cell-type-specific responses to molecular cues in the basal lamina on which they sit. Different regions of the lamina are rich in different types of laminin: α1 and α2 laminin subunits are concentrated in the basal lamina of the crypts, while α5 is in the basal lamina of the villi, in a concentration gradient with its maximum at the villus tip.

Components of the Wnt Signaling Pathway Are Required to Maintain the Gut Stem-Cell Population

What controls whether a gut stem cell retains its stem-cell character or embarks on differentiation? What drives the diversification of the stem cell progeny to produce four different cell types? Despite their importance in normal life and in many diseases, including common forms of cancer, the answers are unknown. There are, however, some clues.

The production of enteroendocrine cells, for example, depends on a cell-fate choice governed by the Notch signaling pathway in much the same way as the production of neurons in the embryonic central nervous system (discussed in Chapter 21). Mutations that block Notch signaling cause enteroendocrine cells to be produced in excess at the expense of other cell types, at least in the embryo.

Other experiments indicate that the Wnt signaling pathway has a crucial role in maintaining the stem cell population of the gut. Mice deficient in one of the LEF-1/TCF family of gene regulatory proteins involved in Wnt signaling (discussed in Chapter 15) provide one line of evidence: villi covered with differentiated cells are formed in the mutant fetus, but the epithelium cannot be renewed, because no crypts form and no proliferating stem cells are retained, and the mouse dies soon after birth. Conversely, in adult life, overactivation of the same signaling pathway by mutations in another gene (the APC gene—see Figure 15-72) result in an overproliferation of crypt cells and frequently lead to cancer, as we shall see in Chapter 23. All this suggests that in the gut, as in the epidermis, Wnt signaling has a key role in either maintaining stem cells as stem cells or controlling their proliferation.

The Liver Functions as an Interface Between the Digestive Tract and the Blood

As we have just seen, the functions of the gut are divided between a variety of cell types. Some cells are specialized for the secretion of hydrochloric acid, others for the secretion of enzymes, others for the absorption of nutrients, and so on. Some of these cell types are closely intermingled in the wall of the gut, whereas others are segregated in large glands that communicate with the gut and originate in the embryo as outgrowths of the gut epithelium.

The liver is the largest of these glands. It develops at a site where a major vein runs close to the wall of the primitive gut tube, and the adult organ retains a special relationship with the blood. Cells in the liver that derive from the primitive gut epithelium—the hepatocytes—are arranged in interconnected sheets and cords, with blood-filled spaces called sinusoids running between them (Figure 22-21). The blood is separated from the surface of the hepatocytes by a single layer of flattened endothelial cells that covers the exposed faces of the hepatocytes. This structure facilitates the chief functions of the liver, which center on the exchange of metabolites between hepatocytes and the blood.

The liver is the main site at which nutrients that have been absorbed from the gut and then transferred to the blood are processed for use by other cells of the body. It receives a major part of its blood supply directly from the intestinal tract (via the portal vein). Hepatocytes are responsible for the synthesis, degradation, and storage of a vast number of substances. They play a central part in the carbohydrate and lipid metabolism of the body as a whole, and they secrete most of the protein found in blood plasma. At the same time, the hepatocytes remain connected with the lumen of the gut via a system of minute channels (or canaliculi) and larger ducts (see Figure 22-21B,C) and secrete into the gut by this route both waste products of their metabolism and an emulsifying agent, bile, which helps in the absorption of fats. Hepatocytes are big cells, and about 50% of them (in an adult human) are polyploid, with two, four, eight, or even more times the normal diploid quantity of DNA per cell.

In contrast to the rest of the digestive tract, there seems to be remarkably little division of labor within the population of hepatocytes. Each hepatocyte seems able to perform the same broad range of metabolic and secretory tasks. These fully differentiated cells can also divide repeatedly, when the need arises, as we explain next.

Liver Cell Loss Stimulates Liver Cell Proliferation

The liver illustrates in a striking way one of the great unsolved problems of developmental and tissue biology: what determines the size of an organ of the body, or the quantity of one type of tissue relative to another? For different organs, the answers are almost certainly different, but there is scarcely any case in which the mechanism is well understood.

Hepatocytes normally live for a year or more and are renewed at a slow rate. Even in a slowly renewing tissue, however, a small but persistent imbalance between the rate of cell production and the rate of cell death would lead to disaster. If 2% of the hepatocytes in a human divided each week but only 1% died, the liver would grow to exceed the weight of the rest of the body within 8 years. Homeostatic mechanisms must operate to adjust the rate of cell proliferation and/or the rate of cell death so as to keep the organ at its normal size. This size, moreover, needs to be matched to the size of the rest of the body. Indeed, when the liver of a small dog is grafted into a large dog, it rapidly grows to almost the size appropriate to the host; conversely, when the liver is grafted from a large dog into a small one, it shrinks.

Direct evidence for the homeostatic control of liver cell proliferation comes from experiments in which large numbers of hepatocytes are removed surgically or are intentionally killed by poisoning with carbon tetrachloride. Within a day or so after either sort of damage, a surge of cell division occurs among the surviving hepatocytes, and the lost tissue is quickly replaced. (If the hepatocytes themselves are totally eliminated, another class of cells, located in the bile ducts, can serve as stem cells for the genesis of new hepatocytes, but usually there is no need for this.) If two-thirds of a rat's liver is removed, for example, a liver of nearly normal size can regenerate from the remainder by hepatocyte proliferation within about 2 weeks. Many molecules have been implicated in the triggering of this reaction. One of the most important is a protein called hepatocyte growth factor. It stimulates hepatocytes to divide in culture, and its production increases steeply (by poorly understood mechanisms) in response to liver damage.

The balance between cell births and cell deaths in the adult liver (and other organs too) does not depend exclusively on the regulation of cell proliferation: cell survival controls also play a part. If an adult rat is treated with the drug phenobarbital, for example, hepatocytes are stimulated to divide, causing the liver to enlarge. When the phenobarbital treatment is stopped, hepatocyte cell death greatly increases until the liver returns to its original size, usually within a week or so. The mechanism of this type of cell survival control is unknown, but it has been suggested that hepatocytes, like most vertebrate cells, depend on signals from other cells for their survival and that the normal level of these signals can support only a certain standard number of hepatocytes. If the number of hepatocytes rises above this (as a result of phenobarbital treatment, for example), hepatocyte death will automatically increase to bring their number back down. It is not known how the appropriate levels of survival factors are maintained.

Summary

The lung performs a simple function—gas exchange—but its housekeeping systems are complex. Surfactant-secreting cells help to keep the alveoli from collapsing. Macrophages constantly scour the alveoli for dirt and microorganisms. A mucociliary escalator formed by mucus-secreting goblet cells and beating ciliated cells sweeps debris out of the airways.

In the gut, where more potentially damaging chemical processes occur, the absorptive epithelium is kept in good repair by constant rapid renewal. In the small intestine, stem cells in the crypts generate new absorptive, goblet, enteroendocrine, and Paneth cells, replacing most of the epithelial lining of the intestine every week. The diverse fates of the stem-cell progeny are controlled, in part at least, by the Notch signaling pathway, while the Wnt pathway is required to maintain the stem-cell population.

The liver is a more protected organ, but it too can rapidly adjust its size up or down by cell proliferation or cell death when the need arises. Differentiated hepatocytes remain able to divide throughout life, showing that a specialized class of stem cells is not always needed for tissue renewal.

Endothelial Cells Line All Blood Vessels

The largest blood vessels are arteries and veins, which have a thick, tough wall of connective tissue and and many layers of smooth muscle cells (Figure 22-22). The wall is lined by an exceedingly thin single sheet of endothelial cells, the endothelium, separated from the surrounding outer layers by a basal lamina. The amounts of connective tissue and smooth muscle in the vessel wall vary according to the vessel's diameter and function, but the endothelial lining is always present. In the finest branches of the vascular tree—the capillaries and sinusoids—the walls consist of nothing but endothelial cells and a basal lamina (Figure 22-23), together with a few scattered—but functionally important—pericytes. These are cells of the connective-tissue family, related to vascular smooth muscle cells, that wrap themselves round the small vessels (Figure 22-24).

Thus, endothelial cells line the entire vascular system, from the heart to the smallest capillary, and control the passage of materials—and the transit of white blood cells—into and out of the bloodstream. A study of the embryo reveals, moreover, that arteries and veins develop from small vessels constructed solely of endothelial cells and a basal lamina: pericytes, connective tissue and smooth muscle are added later where required, under the influence of signals from the endothelial cells. The recruitment of pericytes in particular depends on PDGF-B secreted by the endothelial cells, and in mutants lacking this signal protein or its receptor, pericytes in many regions are missing. As a result, the embryonic blood vessels develop microaneurysms—microscopic pathological dilatations—that eventually rupture, as well as other abnormalities, reflecting the importance of signals exchanged in both directions between the pericytes and the endothelial cells.

Once a vessel has matured, signals from the endothelial cells to the surrounding connective tissue and smooth muscle continue to play a crucial part in regulating the vessel's function and structure. For example, the endothelial cells have mechanoreceptors that allow them to sense the shear stress due to flow of blood over their surface; by signaling this information to the surrounding cells, they enable the blood vessel to adapt its diameter and wall thickness to suit the blood flow. Endothelial cells also mediate rapid responses to neural signals for blood vessel dilation, by releasing the gas NO to make smooth muscle relax in the vessel wall, as discussed in Chapter 15.

New Endothelial Cells Are Generated by Simple Duplication of Existing Endothelial Cells

Throughout the vascular system of the adult body, endothelial cells retain a capacity for cell division and movement. If, for example, a part of the wall of the aorta is damaged and denuded of endothelial cells, neighboring endothelial cells proliferate and migrate in to cover the exposed surface.

The proliferation of endothelial cells can be demonstrated by using ³H-thymidine to label cells synthesizing DNA. In most adult tissues, endothelial cells turn over very slowly, with a cell lifetime ranging, for a mouse, from a couple of months (in liver and lung) to years (in brain and muscle). But endothelial cells not only repair and renew the lining of established blood vessels, they also create new blood vessels. They must do this in embryonic tissues to keep pace with growth, in normal adult tissues to support recurrent cycles of remodeling and reconstruction (as, for example, in the lining of the uterus during the menstrual cycle), and in damaged adult tissues to support repair. In such circumstances, they can be roused to proliferate with a doubling time of just a few days. There is some evidence that, where there is a call for rapid blood-vessel growth, the local population of endothelial cells may also increase by recruitment from the blood stream, which has been reported to contain small numbers of endothelial precursor cells derived from the bone marrow.

New Capillaries Form by Sprouting

New vessels in the adult originate as capillaries, which sprout from existing small vessels. This process of angiogenesis occurs in response to specific signals and can be conveniently observed in naturally transparent structures, such as the cornea of the eye. Irritants applied to the cornea induce the growth of new blood vessels from the rim of tissue surrounding the cornea, which has a rich blood supply, in toward the center of the cornea, which normally has none. Thus, the cornea becomes vascularized through an invasion of endothelial cells into the tough collagen-packed corneal tissue.

Observations such as these reveal that endothelial cells that are to form a new capillary grow out from the side of an existing capillary or small venule by extending long pseudopodia, pioneering the formation of a capillary sprout that hollows out to form a tube (Figure 22-25). This process continues until the sprout encounters another capillary, with which it connects, allowing blood to circulate. Endothelial cells on the arterial and venous sides of the developing network of vessels differ in their surface properties, in the embryo at least: the plasma membranes of the arterial cells contain the transmembrane protein ephrin-B2 (see Chapter 15), while the membranes of the venous cells contain the corresponding receptor protein, Eph-B4, which is a receptor tyrosine kinase (discussed in Chapter 15). These molecules mediate a signal delivered at sites of cell-cell contact, and they are essential for the development of a properly organized network of vessels. One suggestion is that they somehow define the rules for joining one piece of growing capillary tube to another.

Experiments in culture show that endothelial cells in a medium containing suitable growth factors will spontaneously form capillary tubes, even if they are isolated from all other types of cells (Figure 22-26). The capillary tubes that develop do not contain blood, and nothing travels through them, indicating that blood flow and pressure are not required for the initiation of a new capillary network.

Angiogenesis Is Controlled by Factors Released by the Surrounding Tissues

Almost every cell, in almost every tissue of a vertebrate, is located within 50–100 μm of a capillary. What mechanism ensures that the system of blood vessels ramifies into every nook and cranny? How is it adjusted so perfectly to the local needs of the tissues, not only during normal development but also in all sorts of pathological circumstances? Wounding, for example, induces a burst of capillary growth in the neighborhood of the damage, to satisfy the high metabolic requirements of the repair process (Figure 22-27). Local irritants and infections also cause a proliferation of new capillaries, most of which regress and disappear when the inflammation subsides. Less benignly, a small sample of tumor tissue implanted in the cornea, which normally lacks blood vessels, causes blood vessels to grow quickly toward the implant from the vascular margin of the cornea; the growth rate of the tumor increases abruptly as soon as the vessels reach it.

In all these cases, the invading endothelial cells respond to signals produced by the tissue that they invade. The signals are complex, but a key part is played by a protein known as vascular endothelial growth factor (VEGF), a distant relative of platelet-derived growth factor (PDGF). The regulation of blood vessel growth to match the needs of the tissue depends on the control of VEGF production, through changes in the stability of its mRNA and in its rate of transcription. The latter control is relatively well understood. A shortage of oxygen, in practically any type of cell, causes an increase in the intracellular concentration of the active form of a gene regulatory protein called hypoxia-inducible factor 1 (HIF-1). HIF stimulates transcription of the VEGF gene (and of other genes whose products are needed when oxygen is in short supply). The VEGF protein is secreted, diffuses through the tissue, and acts on nearby endothelial cells.

The response of the endothelial cells includes at least four components. First, the cells produce proteases to digest their way through the basal lamina of the parent capillary or venule. Second, the endothelial cells migrate toward the source of the signal. Third, the cells proliferate. Fourth, the cells form tubes and differentiate. VEGF acts on endothelial cells selectively to stimulate this entire set of effects. (Other growth factors, including some members of the fibroblast growth factor family, can also stimulate angiogenesis, but they influence other cell types besides endothelial cells.)

As the new vessels form, bringing blood to the tissue, the oxygen concentration rises, HIF-1 activity declines, VEGF production is shut off, and angiogenesis comes to a halt (Figure 22-28). As in all signaling systems, it is as important to switch the signal off correctly as to switch it on. In normal well-oxygenated tissue, the concentration of HIF-1 is kept low by continual degradation of the HIF-1 protein. This degradation depends on ubiquitylation of HIF-1—a process that requires the product of another gene, which is defective in a rare disorder called von Hippel-Lindau (VHL) syndrome. People with this condition are born with only one functional copy of the VHL gene; mutations occurring at random in the body then give rise to cells in which both gene copies are defective. These cells contain large quantities of HIF-1 regardless of oxygen availability, triggering the continual overproduction of VEGF. The result is development of hemangioblastomas, tumors that contain dense masses of blood vessels. The mutant cells that produce the VEGF are apparently themselves encouraged to proliferate by the over-rich nourishment provided by the excess blood vessels, creating a vicious cycle that promotes tumor growth. Loss of the VHL gene product also gives rise to other tumors as well as hemangioblastomas, by mechanisms that may be independent of effects on angiogenesis.

Summary

Endothelial cells form a single cell layer that lines all blood vessels and regulates exchanges between the bloodstream and the surrounding tissues. Signals from endothelial cells organize the growth and development of connective tissue cells that form the surrounding layers of the blood-vessel wall. New blood vessels can develop from the walls of existing small vessels by the outgrowth of endothelial cells, which have the capacity to form hollow capillary tubes even when isolated in culture. Endothelial cells of developing arteries and veins express different cell-surface proteins, which may control the way in which they link up to create a capillary bed.

A homeostatic mechanism ensures that blood vessels permeate every region of the body. Cells that are short of oxygen increase their concentration of hypoxia-inducible factor 1 (HIF-1), which stimulates the production of vascular endothelial growth factor (VEGF). VEGF acts on endothelial cells, causing them to proliferate and invade the hypoxic tissue to supply it with new blood vessels.

The Three Main Categories of White Blood Cells: Granulocytes, Monocytes, and Lymphocytes

All red blood cells belong in a single class, following the same developmental trajectory as they mature, and the same is true of platelets; but there are many distinct types of white blood cells. White blood cells are traditionally grouped into three major categories—granulocytes, monocytes, and lymphocytes—on the basis of their appearance in the light microscope.

Granulocytes contain numerous lysosomes and secretory vesicles (or granules) and are subdivided into three classes according to the morphology and staining properties of these organelles (Figure 22-30). The differences in staining reflect major differences of chemistry and function. Neutrophils (also called polymorphonuclear leucocytes because of their multilobed nucleus) are the most common type of granulocyte; they phagocytose and destroy microorganisms, especially bacteria, and thus have a key role in innate immunity to bacterial infection, as discussed in Chapter 25. Basophils secrete histamine (and, in some species, serotonin) to help mediate inflammatory reactions; they are closely related in function to mast cells, which reside in connective tissues but are also generated from the hemopoietic stem cells. Eosinophils help to destroy parasites and modulate allergic inflammatory responses.

Once they leave the bloodstream, monocytes (see Figure 22-30D) mature into macrophages, which, together with neutrophils, are the main “professional phagocytes” in the body. As discussed in Chapter 13, both types of phagocytic cells contain specialized lysosomes that fuse with newly formed phagocytic vesicles (phagosomes), exposing phagocytosed microorganisms to a barrage of enzymatically produced, highly reactive molecules of superoxide (O₂^-) and hypochlorite (HOCl, the active ingredient in bleach), as well as to a concentrated mixture of lysosomal hydrolases. Macrophages, however, are much larger and longer-lived than neutrophils. They are responsible for recognizing and removing senescent, dead, and damaged cells in many tissues, and they are unique in being able to ingest large microorganisms such as protozoa.

Monocytes also give rise to dendritic cells, such as the Langerhans cells scattered in the epidermis. Like macrophages, dendritic cells are migratory cells that can ingest foreign substances and organisms; but they do not have as active an appetite for phagocytosis and are instead specialized as presenters of foreign antigens to lymphocytes to trigger an immune response. Langerhans cells, for example, ingest foreign antigens in the epidermis and carry these trophies back to present to lymphocytes in lymph nodes.

There are two main classes of lymphocytes, both involved in immune responses: B lymphocytes make antibodies, while T lymphocytes kill virus-infected cells and regulate the activities of other white blood cells. In addition, there are lymphocytelike cells called natural killer (NK) cells, which kill some types of tumor cells and virus-infected cells. The production of lymphocytes is a specialized topic discussed in detail in Chapter 24. Here we shall concentrate mainly on the development of the other blood cells, often referred to collectively as myeloid cells.

The various types of blood cells and their functions are summarized in Table 22-1.

The Production of Each Type of Blood Cell in the Bone Marrow Is Individually Controlled

Most white blood cells function in tissues other than the blood; blood simply transports them to where they are needed. A local infection or injury in any tissue rapidly attracts white blood cells into the affected region as part of the inflammatory response, which helps fight the infection or heal the wound.

The inflammatory response is complex and is mediated by a variety of signal molecules produced locally by mast cells, nerve endings, platelets, and white blood cells, as well as by the activation of complement (discussed in Chapters 24 and 25). Some of these signal molecules act on nearby capillaries, causing the endothelial cells to adhere less tightly to one another but making their surfaces adhesive to passing white blood cells. The white blood cells are thus caught like flies on flypaper and then can escape from the vessel by squeezing between the endothelial cells and crawling across the basal lamina with the aid of digestive enzymes. The initial binding to endothelial cells is mediated by homing receptors called selectins, and the stronger binding required for the white blood cells to crawl out of the blood vessel is mediated by integrins (see Figure 19-30). Other molecules called chemokines are secreted by damaged or inflamed tissue and local endothelial cells; they act as chemoattractants for specific types of white blood cells, causing these cells to become polarized and crawl toward the source of the attractant. As a result, large numbers of white blood cells enter the affected tissue (Figure 22-31).

Other signal molecules produced in the course of an inflammatory response escape into the blood and stimulate the bone marrow to produce more leucocytes and release them into the bloodstream. The bone marrow is the key target for such regulation because, with the exception of lymphocytes and some macrophages, most types of blood cells in adult mammals are generated only in the bone marrow. The regulation tends to be cell-type-specific: some bacterial infections, for example, cause a selective increase in neutrophils, while infections with some protozoa and other parasites cause a selective increase in eosinophils. (For this reason, physicians routinely use differential white blood cell counts to aid in the diagnosis of infectious and other inflammatory diseases.)

In other circumstances erythrocyte production is selectively increased—for example, in the process of acclimatization when one goes to live at high altitude, where oxygen is scarce. Thus, blood cell formation, or hemopoiesis, necessarily involves complex controls, by which the production of each type of blood cell is regulated individually to meet changing needs. It is a problem of great medical importance to understand how these controls operate, and much progress has been made in this area in recent years.

In intact animals, hemopoiesis is more difficult to analyze than is cell turnover in a tissue such as the epidermal layer of the skin. In the epidermis there is a simple, regular spatial organization that makes it easy to follow the process of renewal and to locate the stem cells. This is not true of the hemopoietic tissues. However, hemopoietic cells have a nomadic life-style that makes them more accessible to experimental study in other ways. Dispersed hemopoietic cells are easily obtained and can be readily transferred, without damage, from one animal to another. Moreover, the proliferation and differentiation of individual cells and their progeny can be observed and analyzed in culture, and numerous molecular markers distinguish the various stages of differentiation. Because of this, more is known about the molecules that control blood cell production than about those that control cell production in other mammalian tissues.

Bone Marrow Contains Hemopoietic Stem Cells

The different types of blood cells and their immediate precursors can be recognized in the bone marrow by routine staining methods (Figure 22-32). They are intermingled with one another, as well as with fat cells and other stromal cells (connective-tissue cells), which produce a delicate supporting meshwork of collagen fibers and other extracellular matrix components. In addition, the whole tissue is richly supplied with thin-walled blood vessels, called blood sinuses, into which the new blood cells are discharged. Megakaryocytes are also present; these, unlike other blood cells, remain in the bone marrow when mature and are one of its most striking features, being extraordinarily large (diameter up to 60 μm), with a highly polyploid nucleus. They normally lie close beside blood sinuses, and they extend processes through holes in the endothelial lining of these vessels; platelets pinch off from the processes and are swept away into the blood (Figure 22-33).

Because of the complex arrangement of the cells in bone marrow, it is difficult to identify in ordinary tissue sections any but the immediate precursors of the mature blood cells. The corresponding cells at still earlier stages of development, before any overt differentiation has begun, are confusingly similar in appearance, and although the spatial distribution of cell types has some orderly features, there is no obvious visible characteristic by which the ultimate stem cells can be recognized. To identify and characterize the stem cells, one needs a functional assay, which involves tracing the progeny of single cells. As we shall see, this can be done in vitro simply by examining the colonies that isolated cells produce in culture. The hemopoietic system, however, can also be manipulated so that such clones of cells can be recognized in vivo in the intact animal.

When an animal is exposed to a large dose of x-rays, most of the hemopoietic cells are destroyed and the animal dies within a few days as a result of its inability to manufacture new blood cells. The animal can be saved, however, by a transfusion of cells taken from the bone marrow of a healthy, immunologically compatible donor. Among these cells there are some that can colonize the irradiated host and permanently reequip it with hemopoietic tissue (Figure 22-34). Experiments of this sort prove that the marrow contains hemopoietic stem cells. They also show how one can assay for the presence of hemopoietic stem cells and hence discover the molecular features that distinguish them from other cells.

For this purpose, cells taken from bone marrow are sorted (with the help of a fluorescence-activated cell sorter) according to the surface antigens that they display, and the different fractions are transfused back into irradiated mice. If a fraction rescues an irradiated host mouse, it must contain hemopoietic stem cells. In this way, it has been possible to show that the hemopoietic stem cells are characterized by a specific combination of cell-surface proteins, and by appropriate sorting virtually pure stem cell preparations can be obtained. The stem cells turn out to be a tiny fraction of the bone-marrow population—about 1 cell in 10,000; but this is enough. As few as five such cells injected into a host mouse with defective hemopoiesis are sufficient to reconstitute its entire hemopoietic system, generating a complete set of blood-cell types, as well as fresh stem cells.

A Multipotent Stem Cell Gives Rise to All Classes of Blood Cells

To see what range of cell types a single hemopoietic stem cell can generate, one needs a way of tracing the fate of its progeny. This can be done by marking the individual stem cell genetically, so that the progeny can be identified even after they have been released into the bloodstream. Although several methods have been used for this, a specially engineered retrovirus (a retroviral vector carrying a marker gene) serves the purpose particularly well. The marker virus, like other retroviruses, can insert its own genome into the chromosomes of the cell it infects, but the genes that would enable it to generate new infectious virus particles have been removed. The marker is therefore confined to the progeny of the cells that were originally infected, and the progeny of one such cell can be distinguished from the progeny of another because the chromosomal sites of insertion of the virus are different. To analyze hemopoietic cell lineages, bone marrow cells are first infected with the retroviral vector in vitro and then are transferred into a lethally irradiated recipient; DNA probes can then be used to trace the progeny of individual infected cells in the various hemopoietic and lymphoid tissues of the host. These experiments show that the individual hemopoietic stem cell is multipotent and can give rise to the complete range of blood cell types, both myeloid and lymphoid, as well as new stem cells like itself (Figure 22-35).

The same methods that were developed for experimentation in mice can now be used for the treatment of disease in humans. Mice, we have seen, can be irradiated to kill off their hemopoietic cells, and then rescued by a transfusion of new stem cells. In the same way, patients with leukemia, for example, can be irradiated or chemically treated to destroy their cancerous cells along with the rest of their hemopoietic tissue, and then can be rescued by a transfusion of hemopoietic stem cells that are free of the cancer-causing mutations. These healthy stem cells can be obtained either from an immunologically matched donor, or by sorting from a sample of bone marrow previously taken from the leukemic patient himself or herself.

The same technology also opens the way, in principle, to one form of gene therapy: hemopoietic stem cells can be isolated in culture, genetically modified by DNA transfection or some other technique to introduce a desired gene, and then transfused back into a patient in whom the gene was lacking, to provide a self-renewing source of the missing genetic component.

Commitment Is a Stepwise Process

Hemopoietic stem cells do not jump directly from a multipotent state into a commitment to just one pathway of differentiation; instead, they go through a series of progressive restrictions. The first step is commitment to either a myeloid or a lymphoid fate. This is thought to give rise to two kinds of progenitor cells, one capable of generating large numbers of all the different types of myeloid cells, or perhaps of myeloid cells plus B lymphocytes, and the other to large numbers of all the different types of lymphoid cells, or at least T lymphocytes. Further steps give rise to progenitors committed to the production of just one cell type. The steps of commitment can be correlated with changes in the expression of specific gene regulatory proteins, needed for the production of different subsets of blood cells. These seem to act in a complicated combinatorial fashion: the GATA-1 protein, for example, is needed for the maturation of red blood cells, but is active also at much earlier steps in the hemopoietic pathway.

The meaning of “commitment” in molecular terms is still unclear, but it has at least two aspects: genes for the chosen mode of differentiation begin to be switched on, while access to other developmental pathways is shut off. These processes normally go hand-in-hand, but studies of mutants show that they are in principle distinct. Animals lacking the gene regulatory protein Pax5 have a block in the production of mature B lymphocytes: progenitor cells begin the process leading to B cell restriction but do not complete it. Normal progenitors that have taken the same initial step cannot be induced, no matter what their environment, to differentiate along any other pathway than that of a B lymphocyte. But the Pax5-defective cells show no such restriction: exposure to appropriate conditions can drive them to generate other blood cell types, including T lymphocytes, macrophages, and granulocytes. This is because the Pax5 protein is required not only to activate genes required for B cell development, but also to shut off genes required for development along other blood-cell pathways.

The Number of Specialized Blood Cells Is Amplified by Divisions of Committed Progenitor Cells

Hemopoietic progenitor cells generally become committed to a particular pathway of differentiation long before they cease proliferating and terminally differentiate. The committed progenitors go through many rounds of cell division to amplify the ultimate number of cells of the given specialized type. In this way, a single stem-cell division can lead to the production of thousands of differentiated progeny, which explains why the number of stem cells is so small a fraction of the total population of hemopoietic cells. For the same reason, a high rate of blood cell production can be maintained even though the stem-cell division rate is low. Infrequent division is a common feature of stem cells in several tissues, including epidermis and gut, as well as the hemopoietic system. The amplifying divisions of the committed progenitors reduce the number of division cycles that the stem cells themselves have to undergo in the course of a lifetime, thereby reducing the risk of replicative senescence (discussed in Chapter 17) and damaging mutations.

The stepwise nature of commitment means that the hemopoietic system can be viewed as a hierarchical family tree of cells. Multipotent stem cells give rise to committed progenitor cells, which are specified to give rise to only one or a few blood cell types. The committed progenitors divide rapidly, but only a limited number of times, before they terminally differentiate into cells that divide no further and die after several days or weeks. Many cells normally die at the earlier steps in the pathway as well. Studies in culture provide a way to find out how the proliferation, differentiation, and death of the hemopoietic cells are regulated.

Stem Cells Depend on Contact Signals From Stromal Cells

Hemopoietic cells can survive, proliferate, and differentiate in culture if, and only if, they are provided with specific signal proteins or accompanied by cells that produce these proteins. If deprived of such proteins, the cells die. For long-term maintenance, contact with appropriate supporting cells also seems to be necessary: hemopoiesis can be kept going for months or even years in vitro by culturing dispersed bone-marrow hemopoietic cells on top of a layer of bone-marrow stromal cells, which mimic the environment in intact bone marrow. Such cultures can generate all the types of myeloid cells, and their long-term continuation implies that stem cells, as well as differentiated progeny, are being continually produced.

There has to be some mechanism, in these cultures as well as in vivo, to guarantee that while some stem cell progeny become committed to differentiation, others remain as stem cells. The cell-fate choice seems to be controlled, in part at least, by specific signals generated at contacts with stromal cells. These signals seem to be needed by the hemopoietic stem cells to keep them in their uncommitted state, just as signals from contacts with the basal lamina are needed to maintain stem cells of the epidermis. In both systems, stem cells are normally confined to a particular niche, and when they lose contact with this niche, they tend to lose their stem-cell potential (Figure 22-36).

The nature of the critical signals from the stromal cells is not yet certain, although several mechanisms have been implicated, involving both secreted and cell-surface-attached factors. For example, hemopoietic precursor cells, including stem cells, in the bone marrow display the transmembrane receptor Notch1 in their plasma membrane, while marrow stromal cells express Notch ligands, and there is evidence that Notch activation helps to keep the stem cells or progenitor cells from embarking on differentiation (see Chapter 15).

Another contact interaction that is important for the maintenance of hemopoiesis came to light through the analysis of mouse mutants with a curious combination of defects: a shortage of red blood cells (anemia), of germ cells (sterility), and of pigment cells (white spotting of the skin; see Figure 21-81). As discussed in Chapter 21, this syndrome results from mutations in either of two genes: one, called c-kit, codes for a receptor tyrosine kinase; the other, called Steel, codes for its ligand, stem-cell factor (SCF). The cell types affected by the mutations all derive from migratory precursors, and it seems that these precursors in each case must express the receptor (Kit) and be provided with the ligand (SCF) by their environment if they are to survive and produce progeny in normal numbers. Studies in mutant mice suggest that SCF must be membrane-bound to be effective, implying that normal hemopoiesis requires direct cell-cell contact between the hemopoietic cells that express Kit receptor protein and stromal cells that express SCF.

Factors That Regulate Hemopoiesis Can Be Analyzed in Culture

While stem cells depend on contact with stromal cells for long-term maintenance, their committed progeny do not, or not to the same degree. Thus, dispersed bone-marrow hemopoietic cells can be cultured in a semisolid matrix of dilute agar or methylcellulose, and factors derived from other cells can be added artificially to the medium. Because cells in the semisolid matrix cannot migrate, the progeny of each isolated precursor cell remain together as an easily distinguishable colony. A single committed neutrophil progenitor, for example, may give rise to a clone of thousands of neutrophils. Such culture systems have provided a way to assay for the factors that support hemopoiesis and hence to purify them and explore their actions. These substances are found to be glycoproteins and are usually called colony-stimulating factors (CSFs). Of the growing number of CSFs that have been defined and purified, some circulate in the blood and act as hormones, while others act in the bone marrow either as secreted local mediators or, like SCF, as membrane-bound signals that act through cell-cell contact. The best understood of the CSFs that act as hormones is the glycoprotein erythropoietin, which is produced in the kidneys and regulates erythropoiesis, the formation of red blood cells.

Erythropoiesis Depends on the Hormone Erythropoietin

The erythrocyte is by far the most common type of cell in the blood (see Table 22-1). When mature, it is packed full of hemoglobin and contains practically none of the usual cell organelles. In an erythrocyte of an adult mammal, even the nucleus, endoplasmic reticulum, mitochondria, and ribosomes are absent, having been extruded from the cell in the course of its development (Figure 22-37). The erythrocyte therefore cannot grow or divide; the only possible way of making more erythrocytes is by means of stem cells. Furthermore, erythrocytes have a limited life-span—about 120 days in humans or 55 days in mice. Worn-out erythrocytes are phagocytosed and digested by macrophages in the liver and spleen, which remove more than 10¹¹ senescent erythrocytes in each of us each day. Young erythrocytes actively protect themselves from this fate: they have a protein on their surface that binds to an inhibitory receptor on macrophages and thereby prevents their phagocytosis.

A lack of oxygen or a shortage of erythrocytes stimulates cells in the kidney to synthesize and secrete increased amounts of erythropoietin into the bloodstream. The erythropoietin, in turn, stimulates the production of more erythrocytes. Since a change in the rate of release of new erythrocytes into the bloodstream is observed as early as 1–2 days after an increase in erythropoietin levels in the bloodstream, the hormone must act on cells that are very close precursors of the mature erythrocytes.

The cells that respond to erythropoietin can be identified by culturing bone marrow cells in a semisolid matrix in the presence of erythropoietin. In a few days, colonies of about 60 erythrocytes appear, each founded by a single committed erythroid progenitor cell. This cell is known as an erythrocyte colony-forming cell, or CFC-E, (or colony-forming unit, CFU-E) and it gives rise to mature erythrocytes after about six division cycles or less. The CFC-Es do not yet contain hemoglobin, and they are derived from an earlier type of progenitor cell whose proliferation does not depend on erythropoietin. CFC-Es themselves depend on erythropoietin for their survival, as well as for proliferation: if erythropoietin is removed from the cultures, the cells rapidly undergo apoptosis.

A second CSF, called interleukin-3 (IL-3), promotes the survival and proliferation of the earlier erythroid progenitor cells. In its presence, much larger erythroid colonies, each comprising up to 5000 erythrocytes, develop from cultured bone marrow cells in a process requiring a week or 10 days. These colonies derive from erythroid progenitor cells called erythrocyte burst-forming cells, or BFC-Es (or burst-forming units, BFU-E). A BFC-E is distinct from a multipotent stem cell in that it has a limited capacity to proliferate and gives rise to colonies that contain erythrocytes only, even under culture conditions that enable other progenitor cells to give rise to other classes of differentiated blood cells. It is distinct from a CFC-E in that it is insensitive to erythropoietin, and its progeny must go through as many as 12 divisions before they become mature erythrocytes (for which erythropoietin must be present). Thus, the BFC-E is thought to be a progenitor cell committed to erythrocyte differentiation and an early ancestor of the CFC-E.

Multiple CSFs Influence the Production of Neutrophils and Macrophages

The two professional phagocytic cells, neutrophils and macrophages, develop from a common progenitor cell called a granulocyte/macrophage (GM) progenitor cell. Like the other granulocytes (eosinophils and basophils), neutrophils circulate in the blood for only a few hours before migrating out of capillaries into the connective tissues or other specific sites, where they survive for only a few days. They then die by apoptosis and are phagocytosed by macrophages. Macrophages, in contrast, can persist for months or perhaps even years outside the bloodstream, where they can be activated by local signals to resume proliferation.

At least seven distinct CSFs that stimulate neutrophil and macrophage colony formation in culture have been defined, and some or all of these are thought to act in different combinations to regulate the selective production of these cells in vivo. These CSFs are synthesized by various cell types—including endothelial cells, fibroblasts, macrophages, and lymphocytes—and their concentration in the blood typically increases rapidly in response to bacterial infection in a tissue, thereby increasing the number of phagocytic cells released from the bone marrow into the bloodstream. IL-3 is one of the least specific of the factors, acting on multipotent stem cells as well as on most classes of committed progenitor cells, including GM progenitor cells. Various other factors act more selectively on committed GM progenitor cells and their differentiated progeny (Table 22-2), although in many cases they act on certain other branches of the hemopoietic family tree as well.

All of these CSFs, like erythropoietin, are glycoproteins that act at low concentrations (about 10^-12 M) by binding to specific cell-surface receptors, as discussed in Chapter 15. A few of these receptors are transmembrane tyrosine kinases but most belong to the large cytokine receptor family, whose members are usually composed of two or more subunits, one of which is frequently shared among several receptor types (Figure 22-38). The CSFs not only operate on the precursor cells to promote the production of differentiated progeny, they also activate the specialized functions (such as phagocytosis and target-cell killing) of the terminally differentiated cells. Proteins produced artificially from the cloned genes for these factors (sometimes referred to as recombinant proteins because they are made using recombinant DNA technology) are strong stimulators of hemopoiesis in experimental animals. They are now widely used in human patients to stimulate the regeneration of hemopoietic tissue and to boost resistance to infection—an impressive demonstration of how basic cell biological research and animal experiments can lead to better medical treatment.

The Behavior of a Hemopoietic Cell Depends Partly on Chance

Up to this point we have glossed over a central question. CSFs are defined as factors that promote the production of colonies of differentiated blood cells. But precisely what effect does a CSF have on an individual hemopoietic cell? The factor might control the rate of cell division or the number of division cycles that the progenitor cell undergoes before differentiating; it might act late in the hemopoietic lineage to facilitate differentiation; it might act early to influence commitment; or it might simply increase the probability of cell survival (Figure 22-39). By monitoring the fate of isolated individual hemopoietic cells in culture, it has been possible to show that a single CSF, such as GM-CSF, can exert all these effects, although it is still not clear which are most important in vivo.

Studies in vitro indicate, moreover, that there is a large element of chance in the way a hemopoietic cell behaves. At least some of the CSFs seem to act by regulating probabilities, not by dictating directly what the cell shall do. In hemopoietic cell cultures, even if the cells have been selected to be as homogeneous a population as possible, there is a remarkable variability in the sizes and often in the characters of the colonies that develop. And if two sister cells are taken immediately after a cell division and cultured apart under identical conditions, they frequently give rise to colonies that contain different types of blood cells or the same types of blood cells in different numbers. Thus, both the programming of cell division and the process of commitment to a particular path of differentiation seem to involve random events at the level of the individual cell, even though the behavior of the multicellular system as a whole is regulated in a reliable way.

Regulation of Cell Survival Is as Important as Regulation of Cell Proliferation

While such observations show that CSFs are not strictly required to instruct the hemopoietic cells how to differentiate or how many times to divide, CSFs are required to keep the cells alive: the default behavior of the cells in the absence of CSFs is death by apoptosis (discussed in Chapter 17). In principle, the CSFs could regulate the numbers of the various types of blood cells entirely through selective control of cell survival in this way, and there is increasing evidence that the control of cell survival plays a central part in regulating the numbers of blood cells, just as it does for hepatocytes and many other cell types, as we have already seen. The amount of apoptosis in the vertebrate hemopoietic system is enormous: billions of neutrophils die in this way each day in an adult human, for example. Indeed, the vast majority of neutrophils produced in the bone marrow die there without ever functioning. This futile cycle of production and destruction presumably serves to maintain a reserve supply of cells that can be promptly mobilized to fight infection whenever it flares up, or phagocytosed and digested for recycling when all is quiet. Compared with the life of the organism, the lives of cells are cheap.

Too little cell death can be as dangerous to the health of a multicellular organism as too much proliferation. In the hemopoietic system, mutations that inhibit cell death by causing excessive production of the intracellular apoptosis inhibitor Bcl-2 promote the development of cancer in B lymphocytes. Indeed, the capacity for unlimited self-renewal is a dangerous property for any cell to possess, and many cases of leukemia arise through mutations that confer this capacity on committed hemopoietic precursor cells that would normally be fated to differentiate and die after a limited number of division cycles.

Summary

The many types of blood cells, including erythrocytes, lymphocytes, granulocytes, and macrophages, all derive from a common multipotent stem cell. In the adult, hemopoietic stem cells are found mainly in bone marrow, and they depend on contact-mediated signals from the marrow stromal (connective-tissue) cells to maintain their stem-cell character. The stem cells normally divide infrequently to produce more stem cells (self-renewal) and various committed progenitor cells (transit amplifying cells), each able to give rise to only one or a few types of blood cells. The committed progenitor cells divide extensively under the influence of various protein signal molecules (colony-stimulating factors, or CSFs) and then terminally differentiate into mature blood cells, which usually die after several days or weeks.

Studies of hemopoiesis have been greatly aided by in vitro assays in which stem cells or committed progenitor cells form clonal colonies when cultured in a semisolid matrix. The progeny of stem cells seem to make their choices between alternative developmental pathways in a partly random manner. Cell death by apoptosis, controlled by the availability of CSFs, also plays a central part in regulating the numbers of mature differentiated blood cells.

New Skeletal Muscle Fibers Form by the Fusion of Myoblasts

The previous chapter described how certain cells, originating from the somites of a vertebrate embryo at a very early stage, become determined as myoblasts, the precursors of skeletal muscle fibers. As discussed in Chapter 7, the commitment to be a myoblast depends on gene regulatory proteins of at least two families—the MyoD family of basic helix-loop-helix proteins, and the MEF2 family of MADS box proteins. These act in combination to give the myoblast a memory of its committed state, and, eventually, to regulate the expression of other genes that give the mature muscle cell its specialized character (see Figure 7-72). After a period of proliferation, the myoblasts undergo a dramatic switch of phenotype that depends on the coordinated activation of a whole battery of muscle-specific genes, a process known as myoblast differentiation. As they differentiate, they fuse with one another to form multinucleate skeletal muscle fibers (Figure 22-41). Fusion involves specific cell-cell adhesion molecules that mediate recognition between newly differentiating myoblasts and fibers. Once differentiation has occurred, the cells do not divide and the nuclei never again replicate their DNA.

Myoblasts that have been kept proliferating in culture for as long as two years still retain the ability to differentiate and can fuse to form muscle cells in response to a suitable change in culture conditions. Appropriate signal proteins such as fibroblast or hepatocyte growth factor (FGF or HGF) in the culture medium can maintain myoblasts in the proliferative, undifferentiated state: if these soluble factors are removed, the cells rapidly stop dividing, differentiate, and fuse. The system of controls is complex, however, and attachment to the extracellular matrix is also important for myoblast differentiation. Moreover, the process of differentiation is cooperative: differentiating myoblasts secrete factors that apparently encourage other myoblasts to differentiate. In the intact animal, the myoblasts and muscle fibers are held in the meshes of a connective-tissue framework formed by fibroblasts. This framework guides muscle development and controls the arrangement and orientation of the muscle cells.

Muscle Cells Can Vary Their Properties by Changing the Protein Isoforms They Contain

Once formed, a skeletal muscle fiber grows, matures, and modulates its character according to functional requirements. The genome contains multiple variant copies of the genes encoding many of the characteristic proteins of the skeletal muscle cell, and the RNA transcripts of many of these genes can be spliced in several ways. As a result, a wealth of protein variants (isoforms) can be produced for the components of the contractile apparatus. As the muscle fiber matures, different isoforms are produced, adapted to the changing demands for speed, strength, and endurance in the fetus, the newborn, and the adult. Within a single adult muscle, several distinct types of skeletal muscle fibers, each with different sets of protein isoforms and different functional properties, can be found side by side (Figure 22-42). Slow muscle fibers (for sustained contraction) and fast muscle fibers (for rapid twitch) are innervated by slow and fast motor neurons, respectively, and the innervation can regulate muscle-fiber gene expression and size through the different patterns of electrical stimulation that these neurons deliver.

Skeletal Muscle Fibers Secrete Myostatin to Limit Their own Growth

A muscle can grow in three ways: its fibers can increase in number, in length, or in girth. Because skeletal muscle fibers are unable to divide, more of them can be made only by the fusion of myoblasts, and the adult number of multinucleated skeletal muscle fibers is in fact attained early—before birth, in humans. Once formed, a skeletal muscle fiber generally survives for the entire lifetime of the animal. However, individual muscle nuclei can be added or lost. Thus, the enormous postnatal increase in muscle bulk is achieved by cell enlargement. Growth in length depends on recruitment of more myoblasts into the existing multinucleated fibers, which increases the number of nuclei in each cell. Growth in girth, such as occurs in the muscles of weightlifters, involves both myoblast recruitment and an increase in the size and numbers of the contractile myofibrils that each muscle fiber nucleus supports.

What, then, are the mechanisms that control muscle cell numbers and muscle cell size? One part of the answer lies in an extracellular signal protein called myostatin. Mice with a loss-of-function mutation in the myostatin gene have enormous muscles—two to three times larger than normal (Figure 22-43). Both the numbers and the size of the muscle cells seem to be increased. Mutations in the same gene turn out to be present in so-called “double-muscled” breeds of cattle (see Figure 17-51): in selecting for big muscles, cattle breeders have unwittingly selected for myostatin deficiency. Myostatin belongs to the TGFβ superfamily of signal proteins, and it is normally made and secreted by skeletal muscle cells. Its function, evidently, is to provide negative feedback to limit muscle growth. Small amounts of the protein can be detected in the circulation of adult humans, and it has been reported that the amount is raised in AIDS patients who show muscle wasting. Thus, myostatin may act as a negative regulator of muscle growth in adult life as well as during development. The growth of some other organs is similarly controlled by a negative-feedback action of a factor that they themselves produce. We shall encounter another example in a later section.

Some Myoblasts Persist as Quiescent Stem Cells in the Adult

Even though humans do not normally generate new skeletal muscle fibers in adult life, the capacity for doing so is not completely lost. Cells capable of serving as myoblasts are retained as small, flattened, and inactive cells lying in close contact with the mature muscle cell and contained within its sheath of basal lamina (Figure 22-44). If the muscle is damaged, these satellite cells are activated to proliferate, and their progeny can fuse to repair the damaged muscle. Satellite cells are thus the stem cells of adult skeletal muscle, normally held in reserve in a quiescent state but available when needed as a self-renewing source of terminally differentiated cells. Athletes who specialize in muscular strength often damage their muscle fibers and are thought to depend on this mechanism for muscle repair, resulting in regenerated fibers that are often highly branched.

The process of muscle repair by means of satellite cells is, nevertheless, limited in what it can achieve. In one form of muscular dystrophy, for example, differentiated skeletal muscle cells are damaged because of a genetic defect in the cytoskeletal protein dystrophin. As a result, satellite cells proliferate to repair the damaged muscle fibers. This regenerative response is, however, unable to keep pace with the damage, and the muscle cells are eventually replaced by connective tissue, blocking any further possibility of regeneration. A similar loss of capacity for repair seems to contribute to the weakening of muscle in the elderly.

In muscular dystrophy, where the satellite cells are constantly called upon to proliferate, their capacity to divide may become exhausted as a result of progressive shortening of their telomeres in the course of each cell cycle (discussed in Chapter 17). Stem cells of other tissues, such as blood, are limited in the same way: they normally divide only at a slow rate, and mutations or exceptional circumstances that cause them to divide more rapidly can lead to premature exhaustion of the stem-cell supply.

Summary

Skeletal muscle fibers are one of the four main categories of vertebrate cells specialized for contraction, and they are responsible for all voluntary movement. Each skeletal muscle fiber is a syncytium and develops by the fusion of many myoblasts. Myoblasts proliferate extensively, but once they have fused, they can no longer divide. Fusion generally follows the onset of myoblast differentiation, in which many genes encoding muscle-specific proteins are switched on coordinately. Some myoblasts persist in a quiescent state as satellite cells in adult muscle; when a muscle is damaged, these cells are reactivated to proliferate and to fuse to replace the muscle cells that have been lost. Muscle bulk is regulated homeostatically by a negative-feedback mechanism, in which existing muscle secretes myostatin, which inhibits further muscle growth.

Fibroblasts Change Their Character in Response to Chemical Signals

Fibroblasts seem to be the least specialized cells in the connective-tissue family. They are dispersed in connective tissue throughout the body, where they secrete a nonrigid extracellular matrix that is rich in type I and/or type III collagen, as discussed in Chapter 19. When a tissue is injured, the fibroblasts nearby proliferate, migrate into the wound, and produce large amounts of collagenous matrix, which helps to isolate and repair the damaged tissue. Their ability to thrive in the face of injury, together with their solitary lifestyle, may explain why fibroblasts are the easiest of cells to grow in culture—a feature that has made them a favorite subject for cell biological studies (Figure 22-46).

As indicated in Figure 22-45, fibroblasts also seem to be the most versatile of connective-tissue cells, displaying a remarkable capacity to differentiate into other members of the family. There are uncertainties about their interconversions, however. Strong evidence indicates that fibroblasts in different parts of the body are intrinsically different, and there may be differences between them even in a single region. “Mature” fibroblasts with a lesser capacity for transformation may, for example, exist side by side with “immature” fibroblasts (often called mesenchymal cells) that can develop into a variety of mature cell types.

The stromal cells of bone marrow, mentioned earlier, provide a good example of connective-tissue versatility. These cells, which can be regarded as a kind of fibroblast, can be isolated from the bone marrow and propagated in culture. Large clones of progeny can be generated in this way from single ancestral stromal cells. According to the signal proteins that are added to the culture medium, the members of such a clone can either continue proliferating to produce more cells of the same type, or can differentiate as fat cells, cartilage cells, or bone cells. Because of their self-renewing, multipotent character, they are referred to as mesenchymal stem cells.

Fibroblasts from the skin are different. Placed in the same culture conditions, they do not show the same plasticity. Yet they, too, can be induced to change their character. At a healing wound, for example, they change their actin gene expression and take on some of the contractile properties of smooth muscle cells, thereby helping to pull the wound margins together; such cells are called myofibroblasts. More dramatically, if a preparation of bone matrix, made by grinding bone into a fine powder and dissolving away the hard mineral component, is implanted in the dermal layer of the skin, some of the cells there (probably fibroblasts) become transformed into cartilage cells, and a little later, others transform into bone cells, thereby creating a small lump of bone. These experiments suggest that components in the extracellular matrix can dramatically influence the differentiation of connective-tissue cells.

We shall see that similar cell transformations are important in the natural repair of broken bones. In fact, bone matrix contains high concentrations of several signal proteins that can affect the behavior of connective-tissue cells. These include members of the TGFβ superfamily, including BMPs and TGFβ itself. These factors are powerful regulators of growth, differentiation, and matrix synthesis by connective-tissue cells, exerting a variety of actions depending on the target cell type and the combination of other factors and matrix components that are present. When injected into a living animal, they can induce the formation of cartilage, bone, or fibrous matrix, according to the site and circumstances of injection. TGFβ is especially important in wound healing, where it stimulates conversion of fibroblasts into myofibroblasts and promotes formation of the collagen-rich scar tissue that gives a healed wound its strength.

The Extracellular Matrix May Influence Connective-Tissue Cell Differentiation by Affecting Cell Shape and Attachment

The extracellular matrix may influence the differentiated state of connective-tissue cells through physical as well as chemical effects. This has been shown in studies on cultured cartilage cells, or chondrocytes. Under appropriate culture conditions, these cells proliferate and maintain their differentiated character, continuing for many cell generations to synthesize large quantities of highly distinctive cartilage matrix, with which they surround themselves. If, however, the cells are kept at relatively low density and remain as a monolayer on the culture dish, a transformation occurs. They lose their characteristic rounded shape, flatten down on the substratum, and stop making cartilage matrix: they stop producing type II collagen, which is characteristic of cartilage, and start producing type I collagen, which is characteristic of fibroblasts. By the end of a month in culture, almost all the cartilage cells have switched their collagen gene expression and taken on the appearance of fibroblasts. The biochemical change must occur abruptly, since very few cells are observed to make both types of collagen simultaneously.

Several lines of evidence suggest that the biochemical change is induced at least in part by the change in cell shape and attachment. Cartilage cells that have made the transition to a fibroblastlike character, for example, can be gently detached from the culture dish and transferred to a dish of agarose. By forming a gel around them, the agarose holds the cells suspended without any attachment to a substratum, forcing them to adopt a rounded shape. In these circumstances, the cells promptly revert to the character of chondrocytes and start making type II collagen again. Cell shape and anchorage may control gene expression through intracellular signals generated at focal contacts by integrins acting as matrix receptors, as discussed in Chapter 19.

For most types of cells, and especially for a connective-tissue cell, the opportunities for anchorage and attachment depend on the surrounding matrix, which is usually made by the cell itself. Thus, a cell can create an environment that then acts back on the cell to reinforce its differentiated state. Furthermore, the extracellular matrix that a cell secretes forms part of the environment for its neighbors as well as for the cell itself, and thus tends to make neighboring cells differentiate in the same way (see Figure 19-61). A group of chondrocytes forming a nodule of cartilage, for example, either in the developing body or in a culture dish, can be seen to enlarge by the conversion of neighboring fibroblasts into chondrocytes.

Fat Cells Can Develop From Fibroblasts

Fat cells, or adipocytes, also derive from fibroblastlike cells, both during normal mammalian development and in various pathological circumstances. In muscular dystrophy, for example, where the muscle cells die, they are gradually replaced by fatty connective tissue, probably by conversion of local fibroblasts. Fat cell differentiation (whether normal or pathological) begins with the expression of two families of gene regulatory proteins: the C/EBP (CCAAT/enhancer binding protein) family and the PPAR (peroxisome proliferator-activated receptor) family, especially PPARγ. Like the MyoD and MEF2 families in skeletal muscle development, the C/EBP and PPARγ proteins drive and maintain one another's expression, through various cross-regulatory and autoregulatory control loops. They work together to control the expression of the other genes characteristic of adipocytes.

The production of enzymes for import of fatty acids and glucose and for fat synthesis leads to an accumulation of fat droplets, consisting mainly of triacylglycerol (see Figure 2-77). These then coalesce and enlarge until the cell is hugely distended (up to 120 μm in diameter), with only a thin rim of cytoplasm around the mass of lipid (Figures 22-47 and 22-48). Lipases are also made in the fat cell, giving it the capacity to reverse the process of lipid accumulation, by breaking down the triacylglycerols into fatty acids that can be secreted for consumption by other cells. The fat cell can change its volume by a factor of a thousand as it accumulates and releases lipid.

Leptin Secreted by Fat Cells Provides Negative Feedback to Inhibit Eating

For almost all animals under natural circumstances, food supplies are variable and unpredictable. Fat cells have the vital role of storing reserves of nourishment in times of plenty and releasing them in times of dearth. It is thus essential to the function of adipose tissue that its quantity should be adjustable throughout life, according to the supply of nutrients. For our ancestors, this was a blessing; in the well-fed half of the modern world, it has become also a curse. In the United States, for example, it is estimated that more than 30% of the population suffer from obesity, defined as a body mass index (weight/height²) more than 30 kg/m², equivalent to about 30% above ideal weight.

It is not easy to determine to what extent the changes in the quantity of adipose tissue depend on changes in the numbers of fat cells, as opposed to changes in fat-cell size. Changes in cell size are probably the main factor in normal nonobese adults, but in severe obesity, at least, the number of fat cells also increases. The factors that drive the recruitment of new fat cells are not well understood, although they are thought to include growth hormone and IGF-1 (insulinlike growth factor-1). It is clear, however, that the increase or decrease of fat cell size is regulated directly by levels of circulating nutrients and by hormones, such as insulin, that reflect nutrient levels. The surplus of food intake over energy expenditure thus directly governs the accumulation of adipose tissue.

But how are food intake and energy expenditure themselves regulated? A human adult eats about a million kilocalories per year, equivalent to about 200 kg of pure fat. Clearly, if we are not to get hopelessly fat or hopelessly thin over the course of a lifetime, there must be control mechanisms to adjust our eating and energy expenditure over the long term according to the quantity of our fat reserves. The key signal is a protein hormone called leptin, which circulates in the bloodstream. Mutant mice that lack leptin or the appropriate leptin receptor are extremely fat (Figure 22-49). Mutations in the same genes sometimes occur in humans, although very rarely. The consequences are similar: constant hunger, overeating, and crippling obesity.

Leptin is normally made by fat cells; the bigger they are, the more they make. Leptin acts on many tissues, and in particular in the brain, on cells in those regions of the hypothalamus that regulate eating behavior. The effect in the brain is to lessen hunger and discourage eating, which results in a decreased amount of fat tissue. Thus, leptin, like myostatin released from muscle cells, provides a negative-feedback mechanism to regulate the growth of the tissue that secretes it.

In most obese people, leptin levels in the blood stream are persistently high. Although leptin receptors are present and functional, the effect of leptin on food intake is overwhelmed by other influences, which are poorly understood.

Bone Is Continually Remodeled by the Cells Within It

Bone is a very dense, specialized form of connective tissue, as different as could be from adipose tissue, even though closely related in origin. Like reinforced concrete, bone matrix is predominantly a mixture of tough fibers (type I collagen fibrils), which resist pulling forces, and solid particles (calcium phosphate as hydroxyapatite crystals), which resist compression. The volume occupied by the collagen is nearly equal to that occupied by the calcium phosphate. The collagen fibrils in adult bone are arranged in regular plywoodlike layers, with the fibrils in each layer lying parallel to one another but at right angles to the fibrils in the layers on either side.

For all its rigidity, bone is by no means a permanent and immutable tissue. Running through the hard extracellular matrix are channels and cavities occupied by living cells, which account for about 15% of the weight of compact bone. These cells are engaged in an unceasing process of remodeling: one class of cells (osteoclasts, related to macrophages) demolishes old bone matrix while another (osteoblasts, related to fibroblasts) deposits new bone matrix. This mechanism provides for continuous turnover and replacement of the matrix in the interior of the bone.

Unlike soft tissues, which can grow by internal expansion, bone can grow only by apposition—that is, by the laying down of additional matrix and cells on the free surfaces of existing bone. During development, this process must occur in coordination with the growth of other tissues, in such a way that the pattern of the body can be scaled up without its proportions being radically disturbed. For most of the skeleton, and in particular for the long bones of the limbs and trunk, the coordinated growth is achieved by a complex strategy. A set of minute “scale models” of these bones are first formed out of cartilage. Each scale model then grows, and as new cartilage is formed, the older cartilage is replaced by bone. Cartilage growth and erosion and bone deposition are so ingeniously coordinated during development that the adult bone, though it may be half a meter long, is almost the same shape as the initial cartilaginous model, which was no more than a few millimeters long.

Defective growth of cartilage during the development of long bones, as a result of a dominant mutation in the gene that codes for an FGF receptor (FGFR3), is responsible for the commonest form of dwarfism, known as achondroplasia (Figure 22-50). Conversely, osteoblasts are lacking in individuals with mutations that disrupt production of a gene regulatory protein (called CBFA1) specifically required for osteoblast differentiation: mice homozygous for this genetic defect are born with a skeleton consisting solely of cartilage and die soon after birth as a result.

Osteoblasts Secrete Bone Matrix, While Osteoclasts Erode It

Cartilage is a simple tissue, consisting of cells of a single type—chondrocytes—embedded in a more or less uniform matrix. The cartilage matrix is deformable, and the tissue grows by expanding as the chondrocytes divide and secrete more matrix (Figure 22-51). Bone is more complex. The bone matrix is secreted by osteoblasts that lie at the surface of the existing matrix and deposit fresh layers of bone onto it. Some of the osteoblasts remain free at the surface, while others gradually become embedded in their own secretion. This freshly formed material (consisting chiefly of type I collagen) is called osteoid. It is rapidly converted into hard bone matrix by the deposition of calcium phosphate crystals in it. Once imprisoned in hard matrix, the original bone-forming cell, now called an osteocyte, has no opportunity to divide, although it continues to secrete further matrix in small quantities around itself. The osteocyte, like the chondrocyte, occupies a small cavity, or lacuna, in the matrix, but unlike the chondrocyte it is not isolated from its fellows. Tiny channels, or canaliculi, radiate from each lacuna and contain cell processes from the resident osteocyte, enabling it to form gap junctions with adjacent osteocytes (Figure 22-52). Although the networks of osteocytes do not themselves secrete or erode substantial quantities of matrix, they probably play a part in controlling the activities of the cells that do.

While bone matrix is deposited by osteoblasts, it is eroded by osteoclasts (Figure 22-53). These large multinucleated cells originate, like macrophages, from hemopoietic stem cells in the bone marrow. The precursor cells are released as monocytes into the bloodstream and collect at sites of bone resorption, where they fuse to form the multinucleated osteoclasts, which cling to surfaces of the bone matrix and eat it away. Osteoclasts are capable of tunneling deep into the substance of compact bone, forming cavities that are then invaded by other cells. A blood capillary grows down the center of such a tunnel, and the walls of the tunnel become lined with a layer of osteoblasts (Figure 22-54). To produce the plywoodlike structure of compact bone, these osteoblasts lay down concentric layers of new matrix, which gradually fill the cavity, leaving only a narrow canal surrounding the new blood vessel. Many of the osteoblasts become trapped in the bone matrix and survive as concentric rings of osteocytes. At the same time as some tunnels are filling up with bone, others are being bored by osteoclasts, cutting through older concentric systems. The consequences of this perpetual remodeling are beautifully displayed in the layered patterns of matrix observed in compact bone (Figure 22-55).

Through remodelling, bones are endowed with a remarkable ability to adjust their structure in response to long-term variations in the load imposed on them. This adaptive behavior implies that the deposition and erosion of the matrix are somehow controlled by local mechanical stresses, but the mechanisms involved are not understood. The bone cells secrete signal proteins that become trapped in the matrix, and it is likely that these are released when the matrix is degraded or suitably stressed. The released proteins, especially members of the BMP subfamily of TGFβ proteins, may help to guide the remodelling process.

Remodelling carries a risk: defects in its control can lead to osteoporosis, where there is excessive erosion of the bone matrix and weakening of the bone, or to the opposite condition, osteopetrosis, where the bone becomes excessively thick and dense.

During Development, Cartilage Is Eroded by Osteoclasts to Make Way for Bone

The replacement of cartilage by bone in the course of development is also thought to depend on the activities of osteoclasts. As the cartilage matures, its cells in certain regions become greatly enlarged at the expense of the surrounding matrix, and the matrix itself becomes mineralized, like bone, by the deposition of calcium phosphate crystals. The swollen chondrocytes die, leaving large empty cavities. Osteoclasts and blood vessels invade the cavities and erode the residual cartilage matrix, while osteoblasts following in their wake begin to deposit bone matrix. The only surviving remnant of cartilage in the adult long bone is a thin layer that forms a smooth covering on the bone surfaces at joints, where one bone articulates with another (Figure 22-56).

Some cells capable of forming new cartilage persist, however, in the connective tissue that surrounds a bone. If the bone is broken, the cells in the neighborhood of the fracture repair it by a sort of recapitulation of the original embryonic process: cartilage is first laid down to bridge the gap and is then replaced by bone.

The capacity for self-repair, so strikingly illustrated by the tissues of the skeleton, is a property of living structures that has no parallel among present-day man-made objects.

Summary

The family of connective-tissue cells includes fibroblasts, cartilage cells, bone cells, fat cells, and smooth muscle cells. Some classes of fibroblasts seem to be able to transform into any of the other members of the family. These transformations of connective-tissue cell type are regulated by the composition of the surrounding extracellular matrix, by cell shape, and by hormones and growth factors. While the chief function of most members of the family is to secrete extracellular matrix, fat cells serve as storage sites for fat. The quantity of fat tissue is regulated in part by negative feedback: fat cells release a hormone, leptin, which acts in the brain to reduce appetite, which leads to a decrease in fat tissue.

Cartilage and bone both consist of cells embedded in a solid matrix. The matrix of cartilage is deformable so that the tissue can grow by swelling, whereas bone is rigid and can grow only by apposition. Bone undergoes perpetual remodeling through which it can adapt to the load it bears; the remodelling depends on the combined action of osteoclasts, which erode matrix, and osteoblasts, which secrete it. Some osteoblasts become trapped in the matrix as osteocytes and play a part in regulating the turnover of bone matrix. Most long bones develop from miniature cartilage “models,” which, as they grow, serve as templates for the deposition of bone by the combined action of osteoblasts and osteoclasts. Similarly, in the repair of a bone fracture in the adult, the gap is first bridged by cartilage, which is later replaced by bone.

ES Cells Can Be Used to Make Any Part of the Body

As described in Chapter 21, it is possible through cell culture to derive from early mouse embryos an extraordinary class of stem cells called embryonic stem cells, or ES cells. ES cells can be kept proliferating indefinitely in culture and yet retain an unrestricted developmental potential. If they are put back into an early embryonic environment, they can give rise to all the tissues and cell types in the body, including germ cells. They integrate perfectly into whatever site they may come to occupy, adopting the character and behavior that normal cells would show at that site. One can think of development in terms of a series of choices presented to cells as they follow a road that leads from the fertilized egg to terminal differentiation. After their long sojourn in culture, the ES cell and its progeny can evidently still read the signs at each branch in the highway and respond as normal embryonic cells would.

Cells with properties similar to those of mouse ES cells can now be derived from early human embryos and from human fetal ovaries and testes, creating a potentially inexhaustible supply of cells that might be used for the replacement and repair of mature human tissues that are damaged. Whether or not one has ethical objections to such use of human embryos, it is worth considering the possibilities that are opened up. Setting aside the dream of growing entire organs from ES cells by a recapitulation of embryonic development, experiments in mice suggest that it will be possible in the near future to use ES cells to replace the skeletal muscle fibers that degenerate in victims of muscular dystrophy, the nerve cells that die in patients with Parkinson's disease, the insulin-secreting cells that are lacking in type I diabetics, the heart muscle cells that die in a heart attack, and so on.

Mouse ES cells can be induced to differentiate into a variety of cell types in culture (Figure 22-57). When treated with a carefully chosen combination of signal proteins, for example, the ES cells differentiate into astrocytes and oligodendrocytes, the two main types of glial cells in the central nervous system. If the treated ES cells are injected into a mouse brain, they can serve as progenitors of these cell types. If the host mouse is deficient in myelin-forming oligodendrocytes, for example, the grafted cells can correct the deficiency and form myelin sheaths around axons that lack them.

Epidermal Stem Cell Populations Can Be Expanded in Culture for Tissue Repair

It is a long way still from this sort of success in mice to routine treatments for human diseases. One of the main difficulties lies in immune rejection. If ES-derived cells of one genotype are grafted into an individual of another, the grafted cells are likely to be rejected by the immune system as foreign. Methods of dealing with this problem have been developed for the transplantation of organs such as kidneys and hearts. Immunological problems—and some ethical problems—can, however, be avoided altogether if the right kinds of stem cells can be obtained from the patient's own body.

A simple example is the use of epidermal stem cells for repair of the skin after extensive burns. By culturing cells from undamaged regions of the skin of the burned patient, it is possible to obtain epidermal stem cells quite rapidly in large numbers. These can then be used to repopulate the damaged body surface. For good results after a third-degree burn, however, it is essential to provide first of all an urgent replacement for the lost dermis. For this, dermis taken from a human cadaver can be used, or an artificial dermis substitute. This is still an area of active experimentation. In one technique, an artificial matrix of collagen mixed with a glycosaminoglycan is formed into a sheet, with a thin membrane of silicone rubber covering its external surface as a barrier to water loss, and this skin substitute (called Integra) is laid on the burned body surface after the damaged tissue has been cleaned away. Fibroblasts and blood capillaries from the patient's surviving deep tissues migrate into the artificial matrix and gradually replace it with new connective tissue. Meanwhile, the epidermal cells are cultivated until there are enough to form a thin sheet of adequate extent. Two or more weeks after the original operation, the silicone rubber membrane is carefully removed and replaced with this cultured epidermis, so as to reconstruct a complete skin.

Neural Stem Cells Can Repopulate the Central Nervous System

While the epidermis is one of the simplest and most easily regenerated tissues, the central nervous system (the CNS) is the most complex and seems the most difficult to reconstruct in adult life. The adult mammalian brain and spinal cord have very little capacity for self-repair. Stem cells capable of generating new neurons are hard to find in adult mammals—so hard to find, indeed, that until recently they were thought to be absent.

It is now known, however, that CNS neural stem cells capable of giving rise to both neurons and glial cells do persist in the adult mammalian brain. Moreover, in certain parts of the brain they continually produce new neurons to replace those that die (Figure 22-58). Neuronal turnover occurs on a more dramatic scale in certain songbirds, where large numbers of neurons die each year and are replaced by newborn neurons as part of the process by which a new song is learned in each breeding season.

In experiments with rodents, adult neural stem cells have been harvested from the brain, grown in culture, and then implanted back into the brain of a host animal, where they produce differentiated progeny. Remarkably, it seems that the grafted cells adjust their behavior to match their new location. Stem cells from the hippocampus, for example, implanted in the olfactory-bulb-precursor pathway (see Figure 22-58) give rise to neurons that become correctly incorporated into the olfactory bulb, and vice versa. These findings hold out the hope that, in spite of the extraordinary complexity of nerve cell types and neuronal connections, it may be possible to use neural stem cells to repair at least some types of damage and disease in the central nervous system.

The Stem Cells of Adult Tissues May Be More Versatile Than They Seem

Do cells with unexpected stem-cell capabilities lurk in other parts of the body also? If embryonic stem cells can be caused to differentiate along any pathway we please, is it possible to induce other kinds of stem cells, taken from adult tissues, to produce cell types other than their standard range of progeny?

There is now strong evidence that the answer is yes. It has been reported, for example, that neural stem cells, derived from adult brain, can give rise to hemopoietic cells when injected onto a mouse whose own stock of hemopoietic cells has been depleted by x-irradiation. These neural stem cells have also been found to give rise to skeletal muscle cells when injected directly into muscle tissue.

As another important example, cells from adult bone marrow, when injected into x-irradiated recipients, can not only supply the host with fresh hemo-poietic cells, but can also give rise to various other cell types, including pneumocytes in the lung and hepatocytes in the liver. Genesis of liver cells (and some other equally surprising cell types) from bone-marrow cells has been demonstrated both in mice and in humans who have received bone-marrow transplants for the treatment of leukemia. Well-differentiated hepatocytes displaying genetic markers proving that they come from the donor are found in the liver of the host. Experiments in mice have shown that the hepatocytes in such cases derive specifically from the hemopoietic stem cells in the bone marrow.

When the host liver is itself defective or damaged, so as to encourage its repopulation by grafted cells, hepatocytes derived from donor hemopoietic cells can be found in plenty. It is not yet clear, however, how readily the conversion of cell fate takes place. It may be that any given hemopoietic stem cell has only a small probability of switching to an alien fate, even when placed in the appropriate alien environment. As yet, we have only a very hazy idea of which transitions are permitted or what factors provoke or inhibit them.

Stem cells taken directly from adult tissues promise to be useful in many ways for tissue repair, but other strategies may have even greater potential. In principle, at least, it should even be possible to use adult tissues to derive “personalized” ES cells—that is, ES cells with the same genome as the adult patient whose body is in need of repair. The cloning of Dolly the sheep and of other mammals has indicated a way to do this. The nucleus of an egg cell can be artificially replaced by a nucleus derived from an adult cell, and the hybrid cell can then go on to develop into an entire individual whose nuclear genome is identical to that of the adult donor (see Figure 7-2). While most of us would not wish to make human beings in this way, it should be possible to obtain ES cells from the immediate descendants of the hybrid cell, by techniques like those used to derive ES cells from early mouse embryos.

Serious ethical issues to need be resolved and enormous technical problems overcome before such an approach can become a reality. Perhaps other, better ways will be found to restore adult cells to an embryonic state of versatility. But by one route or another, it seems that cell biology is beginning to open up new opportunities for improving on Nature's mechanisms of tissue repair, remarkable as those mechanisms are.

Summary

In the normal adult body, different classes of stem cells are responsible for the renewal of different types of tissue. Some tissues, however, seem incapable of repair by the genesis of new cells because no competent stem cells are present. Recent discoveries have opened up new possibilities for manipulating stem-cell behavior artificially so as to repair tissues that previously seemed unrepairable. Epidermal stem cells taken from undamaged skin of a badly burned patient can be rapidly grown in large numbers in culture and grafted back to reconstruct an epidermis to cover the burns. Neural stem cells persist in a few regions of the adult mammalian brain, and when grafted into a developing or damaged brain can generate new neurons and glia appropriate to the site of grafting.

Embryonic stem cells (ES cells) are able to differentiate into any cell type in the body, and they can be induced to differentiate into many cell types in culture. Stem cells of some adult tissues, such as bone marrow, when placed in a suitable environment, seem able to generate a much wider range of differentiated cell types than they produce normally. These findings of stem-cell biology offer the hope of remedy for many serious diseases.