Chapter 22:  Differentiated Cells and the Maintenance of Tissues

Introduction

Although the tissues of the body differ in many ways, they all have certain basic requirements, usually provided for by a mixture of cell types, as illustrated for the skin in Figure 22-1. They all need mechanical strength, which is often provided by a supporting framework of extracellular matrix, mainly secreted by fibroblasts. In addition, almost all tissues need a blood supply to bring nutrients and remove waste products, and so they are pervaded by blood vessels lined with endothelial cells. Likewise, most tissues are innervated by nerve cell axons, which are ensheathed by Schwann cells. Macrophages are usually present to dispose of dying cells and to remove unwanted extracellular matrix, as are lymphocytes and other white blood cells to combat infection. Melanocytes may be present to provide a protective or decorative pigmentation. Most of these cell types, ancillary to the specialized function of the tissue, originate outside it and invade the tissue either early in the course of its development (endothelial cells, nerve cell axons, Schwann cells, and melanocytes) or continually during life (macrophages and other white blood cells). This complex supporting apparatus is required to maintain the principal specialized cells of the tissue: the contractile cells of the muscle, the secretory cells of the gland, or the blood-forming cells of the bone marrow, for example.

Almost every tissue is therefore an intricate mixture of many cell types that must remain different from one another while coexisting in the same environment. Moreover, the organization of the mixture must be preserved even though, in almost all adult tissues, cells are continually dying and being replaced. The retention of tissue form and function is made possible largely through two fundamental properties of cells. Because of cell memory (see Chapter 21), specialized cells autonomously maintain their distinctive character and pass it on to their progeny. At the same time each type of specialized cell continually senses its environment and adjusts its proliferation and properties to suit the circumstances; in fact, the very survival of most cells depends on signals from other cells. The intracellular mechanisms thought to be responsible for cell memory are discussed in Chapter 9, while the ways in which cells respond to environmental signals are considered in Chapter 15. In this preliminary section on the behavior of cells in tissues, we briefly review some of the evidence for the stability and heritability of the differentiated state and consider to what extent this state can be modified by environmental influences.

Most Differentiated Cells Remember Their Essential Character Even in a Novel Environment ²

Cell culture experiments demonstrate that even when cells are removed from their usual environment, they and their progeny generally remain true to their original instructions. Consider, for example, the epithelial cells that form the pigmented layer of the retina (Figure 22-2). Because they display their specialized character by manufacturing dark brown granules of melanin, it is easy to monitor their state of differentiation. When these cells are isolated from the retina of a chick embryo and grown in culture, they proliferate to form clones. Single cells taken from these clones breed true, giving subclones of similar pigment epithelial cells. The differentiated state can be maintained in this way through more than 50 cell generations.

The behavior of the cells is not, however, independent of their environment. In certain media or in conditions of extreme crowding, they may survive but synthesize little or no pigment. But even when they fail to express their differentiated character, they remain determined as pigment cells: when they are returned to more favorable culture conditions, they synthesize pigment once again. There are one or two known exceptions to this rule. In some vertebrate species, under certain conditions, retinal pigment cells will transdifferentiate into lens cells or into cells of the neural retina, but no manipulation of the conditions has been found to cause them to differentiate instead into blood cells, for example, or into liver cells or heart cells. Similarly, most types of specialized cells, including blood cells, liver cells, and heart cells, maintain their essential character in culture.

In the body, just as in culture, most specialized cells behave as though their basic character has been irreversibly determined by their developmental history. Epidermal cells, for example, remain epidermal cells even in the most alien surroundings: if a suspension of dissociated epidermal cells is prepared from the tail skin of a rat and injected beneath the capsule of the kidney, the cells grow there to form cysts lined with unmistakable epidermis, resembling that on the surface of the body.

The Differentiated State Can Be Modulated by a Cell's Environment ^{1, 3}

Although radical transformations are largely forbidden, the character of many differentiated cells can be strongly influenced by the environment. The possible adjustments can be classified mostly as modulations of the differentiated state - that is, reversible changes between closely related cell phenotypes. Liver cells, for example, adjust their synthesis of specific enzymes (through changes in specific mRNA levels) according to the ambient concentrations of the steroid hormone hydrocortisone, and the production of milk proteins by mammary gland cells can be switched on or off by changes in the extracellular matrix. Fibroblasts and their relatives - the family of connective-tissue cells - are a special case. These cells are exceptionally adaptable and can undergo various interconversions: fibroblasts, for example, can apparently change reversibly into cartilage cells. Such transformations are important in the healing of wounds and bone fractures and in other pathological processes; they are discussed later in this chapter. Even these conversions of one differentiated cell type into another, however, are narrowly restricted: the converted cell remains a member of the family of connective-tissue cells. Important but restricted changes of differentiated state occur also in many normal adult tissues where new differentiated cells are generated from stem cells - precursors that do not themselves display the mature differentiated character but are specialized to divide and to yield progeny that will. Distinct types of stem cells are committed to the production of distinct types of differentiated cells and are not interconvertible.

The majority of adult tissues, therefore, are composed of a number of distinct, irreversibly determined cell lineages. The numbers and spatial relationships of these components have to be maintained throughout life by mechanisms that do not require one type of differentiated cell to transform into another but depend on complex interactions between the different cell types.

Summary

Most differentiated cells in adult tissues will maintain their specialized character even when placed in a novel environment. Although states of differentiation are generally stable and not interconvertible, even highly specialized cells can alter their properties to a limited extent in response to environmental cues. In many adult tissues, moreover, new differentiated cells are continually generated from stem cells that appear undifferentiated. Especially striking cell transformations occur within the family of connective-tissue cells that includes fibroblasts and cartilage cells.

Introduction

Not all the populations of differentiated cells in the body are subject to cell turnover. Some cell types, having been generated in appropriate numbers in the embryo, are retained throughout adult life; they seem never to divide, and they cannot be replaced if they are lost. Almost all nerve cells are permanent in this sense. So are a few other types of cells, including - in mammals - the muscle cells of the heart, the auditory hair cells of the ear (Figure 22-3), and the lens cells of the eye.

While all these cells have extremely long life-spans and necessarily live in protected environments, they are dissimilar in other respects, and it is difficult to give a general reason why they should be permanent and irreplaceable. For heart muscle cells and auditory hair cells it is difficult to give any reason at all. In the case of nerve cells it seems likely that cell turnover in the adult would be disadvantageous as a rule, since it would be difficult to reestablish in the adult the precise and complex patterns of nerve connections that are set up under very different circumstances during development. Moreover, any memories recorded in the form of slight modifications of the structure or interconnections of individual nerve cells would presumably be obliterated. In the lens of the eye, on the other hand, the permanence of the cells appears to be simply an inevitable consequence of the way the tissue grows.

The Cells at the Center of the Lens of the Adult Eye Are Remnants of the Embryo ⁵

Very little of the adult body consists of the same molecules that were laid down in the embryo. The lens of the eye is an exception: it is one of the few structures containing cells that are not only preserved but are preserved without turnover of their contents.

The lens is formed from the ectoderm at the site where the developing optic vesicle makes contact with it: the ectoderm here thickens, invaginates, and finally pinches off as a lens vesicle (see Figure 22-2). The lens thus originates as a spherical shell of cells formed from an epithelium, one cell layer thick, surrounding a central cavity. The cells at the rear of the lens vesicle (those facing the retina) soon undergo a striking transformation. They synthesize and become filled with crystallins, the characteristic proteins of the lens. In the process they elongate enormously, differentiating into lens fibers (Figure 22-4). Eventually, their nuclei disintegrate and protein synthesis ceases. In this way the part of the lens vesicle epithelium facing the retina is expanded into a thick refractile body consisting of many long, lifeless cells packed side by side (Figure 22-5). The central cavity of the vesicle is obliterated, and the front part of the epithelium of the lens vesicle - the part facing the external world - remains as a thin sheet of low cuboidal cells. Growth of the lens depends on the proliferation of these cells at the front, pushing some of the cells from this region around the rim of the lens and toward the back (see Figures 22-4 and 22-5A). As cells move to the rear, they stop dividing, step up their rate of synthesis of crystallins, and differentiate into lens fibers. Additional lens fibers continue to be recruited in this way throughout life, although at an ever decreasing rate.

The types of crystallins filling the earliest generations of lens fibers are different from those of the later generations, just as the hemoglobins of fetal red blood cells are different from those of adult red blood cells. But whereas old red blood cells are discarded, old lens fibers are not. Thus at the core of the adult lens lie fibers that were laid down in the embryo and are still packed with the distinctive types of crystallins manufactured in that earlier period. Differences of refractive index between the early embryonic types of crystallins and those that are laid down later help to free the lens of the eye from the optical aberrations that bedevil simple lenses made out of homogeneous media such as glass.

Most Permanent Cells Renew Their Parts: The Photoreceptor Cells of the Retina ⁶

There are few cells as immutable as lens fibers. As a rule, even those cells that persist throughout life without dividing undergo renewal of their component parts. Thus, while they do not divide, heart muscle cells, auditory hair cells, and nerve cells are metabolically active and capable not only of synthesizing new RNA and protein, but also of altering their size and structure during adult life. Heart muscle cells, for example, replace the bulk of their protein molecules in the course of a week or two, and they will adjust the balance of protein synthesis and degradation so as to grow bigger if the load on the heart is increased - for example, by a sustained increase in blood pressure. Nerve cells also replace their protein molecules continuously; moreover, many nerve cells can regenerate axons and dendrites that have been cut off.

The turnover of cell components is dramatically illustrated in the highly specialized neural cells that form the photoreceptors of the retina. The neural retina (see Figure 22-2) 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 22-6). Rods and cones contain different photosensitive complexes of protein with visual pigment: rods are especially sensitive at low light levels, while cones (of which there are three types, each with different spectral responses) 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 22-7). 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 22-6).

The photoreceptors are permanent cells that do not divide. But the photosensitive protein molecules are not permanent. There is a steady turnover, which can be demonstrated by showing that injected radioactive amino acids are incorporated into these molecules. 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 22-8). 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.

Summary

Some cells in mammals - including nerve cells, heart muscle cells, sensory receptor cells for light and sound, and lens fibers - persist throughout life without dividing and without being replaced. In mature lens fibers the cell nuclei have degenerated and protein synthesis has stopped, so that the core of the adult lens consists of lens proteins laid down early in embryonic life. In most other permanent cells biosynthetic activity continues, and there is a steady turnover of cell components. In the rod cells of the retina, for example, new layers of photoreceptive membrane are synthesized close to the nucleus and are steadily displaced outward until they are eventually engulfed and digested by cells of the pigment epithelium.

Introduction

Most of the differentiated cell populations in a vertebrate are not permanent: the cells are continually dying and being replaced. New differentiated cells can be produced during adult life in either of two ways: (1) they can form by the simple duplication of existing differentiated cells, which divide to give pairs of daughter cells of the same type; or (2) they can be generated from relatively undifferentiated stem cells by a process that involves a change of cell phenotype, as will be explained in detail later in this chapter.

Rates of renewal vary from one tissue to another. The turnover time may be as short as a week or less, as in the epithelial lining of the small intestine (which is renewed by means of stem cells), or as long as a year or more, as in the pancreas (which is renewed by simple duplication). Many tissues whose normal rates of renewal are very slow can be stimulated to produce new cells at higher rates when the need arises.

In this section we discuss two examples of cell populations that are renewed by simple duplication - liver cells and endothelial cells.

The Liver Functions as an Interface Between the Digestive Tract and the Blood ^{7, 8}

Digestion is a complex process. The cells that line the digestive tract secrete into the lumen of the gut a variety of substances, such as hydrochloric acid and digestive enzymes, to break down food molecules into simpler nutrients. The cells absorb these nutrients from the gut lumen, process them, and then release them into the blood for utilization by other cells of the body. All of these activities are adjusted according to the composition of the food consumed and the levels of metabolites in the circulation. The complex set of tasks is performed by a division of labor: some of the cells are specialized for the secretion of hydrochloric acid, others for the secretion of enzymes, others for absorption of nutrients, others for the production of peptide hormones, such as gastrin, that regulate digestive and metabolic activities, and so on (Figure 22-9). Some of these cell types lie closely intermingled in the wall of the gut; 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 singularly close relationship with the blood. The cells in the liver that derive from the primitive gut epithelium - the hepatocytes - are arranged in folded sheets, facing blood-filled spaces called sinusoids (Figure 22-10A). The blood is separated from the surface of the hepatocytes by a single layer of flattened endothelial cells that covers the sides of each hepatocyte sheet (Figure 22-10B). 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 22-10B) 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. 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 appears to be able to perform the same broad range of metabolic and secretory tasks.

Hepatocytes have a life-style different from the cells lining the lumen of the gut. The latter, exposed to the abrasive and corrosive contents of the gut, cannot live for long and must be rapidly replaced by a continual supply of new cells (see Figure 22-17). Hepatocytes, removed from direct contact with the contents of the gut, live much longer and are normally renewed at a slow rate.

Liver Cell Loss Stimulates Liver Cell Proliferation ⁹

Even in a slowly renewing tissue, a small but persistent imbalance between the rate of cell production and the rate of cell death will 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 in order to keep the organ at its standard size.

Direct evidence for 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 two-thirds of a rat's liver is removed, for example, a liver of nearly normal size can regenerate from the remainder within about 2 weeks. In cases of this kind a signal for liver regeneration can be demonstrated in the circulation: if the circulations of two rats are connected surgically and two-thirds of the liver of one of them is excised, cell division is stimulated in the unmutilated liver of the other. One of the signals responsible for the increased cell proliferation has been identified as a protein called hepatocyte growth factor. It stimulates hepatocytes to divide in culture, and its concentration in the bloodstream rises steeply (by poorly understood mechanisms) in response to liver damage. The same factor affects several other cell types in a variety of ways and is also known as scatter factor because it causes some kinds of epithelial cells to become motile so that they dissociate and migrate away from one another. It is not clear why it is specifically the liver that is stimulated to grow after 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 seem also to 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.

Regeneration Requires Coordinated Growth of Tissue Components ¹⁰

Like all organs, the liver comprises a mixture of cell types. Besides the hepatocytes and the endothelial cells that line its sinusoids, it contains both specialized macrophages (Kupffer cells), which engulf particulate matter in the bloodstream and dispose of worn-out red blood cells, and a small number of fibroblasts, which provide a tenuous supporting framework of connective tissue (see Figure 22-10B). All of these cell types are capable of division. For optimal regeneration their proliferation must be properly coordinated.

The importance of balanced regeneration of cell types is demonstrated by what happens when an imbalance occurs. If hepatocytes, for example, are poisoned repeatedly with carbon tetrachloride or with alcohol at such frequent intervals that they cannot recover fully between attacks, the fibroblasts take advantage of the situation and the liver becomes irreversibly clogged with connective tissue, leaving little space for the hepatocytes to grow even after the toxic agents are withdrawn. This condition, called cirrhosis, is common in chronic alcoholics. In a similar way the regeneration of severely damaged skeletal muscle is often seriously hindered by the overgrowth of its connective tissue so that scar tissue replaces the contractile muscle fibers. These imbalances, however, require unusual tissue damage; in ordinary circumstances of tissue renewal, poorly understood mechanisms regulate cell proliferation and cell survival so as to ensure that the proper mixture of cell types is maintained.

Endothelial Cells Line All Blood Vessels ¹¹

By contrast with the above examples of ill-coordinated behavior of fibroblasts, the endothelial cells that form the lining of blood vessels have a remarkable capacity to adjust their number and arrangement to suit local requirements. Almost all tissues depend on a blood supply, and the blood supply depends on endo-thelial cells. They create an adaptable life-support system spreading into almost every region of the body. If it were not for endothelial cells extending and remodeling the network of blood vessels, tissue growth and repair would be impossible.

The largest blood vessels are arteries and veins, which have a thick, tough wall of connective tissue and smooth muscle (Figure 22-11A). The wall is lined by an exceedingly thin single layer of endothelial cells, 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 (Figure 22-11B). 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 22-12). 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: connective tissue and smooth muscle are added later where required, under the influence of signals from the endothelial cells.

New Endothelial Cells Are Generated by Simple Duplication of Existing Endothelial Cells ¹²

Throughout the vascular system of the adult, 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. Newly formed endothelial cells will even cover the inner surface of plastic tubing used by surgeons to replace parts of damaged blood vessels.

The proliferation of endothelial cells can be demonstrated by using ³H-thymidine to label cells synthesizing DNA. In normal vessels the proportion of endothelial cells that become labeled is especially high at branch points in arteries, where turbulence and the resulting wear on the endothelial cells presumably stimulate cell turnover. On the whole, however, endothelial cells turn over very slowly, with a cell lifetime of months or even years.

Endothelial cells not only repair 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, and in damaged adult tissues to support repair.

New Capillaries Form by Sprouting ^{13, 14}

New vessels always originate as capillaries, which sprout from existing small vessels. This process of angiogenesis occurs in response to specific signals. The process can be readily observed in rabbits by making a small hole in the ear and fixing glass coverslips on either side to create a thin transparent viewing chamber into which the cells that surround the wound can grow. Angiogenesis can also 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 the 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 will form a new capillary grow out from the side of a capillary or small venule by extending long processes or pseudopodia (Figure 22-13). The cells at first form a solid sprout, which then hollows out to form a tube. This process continues until the sprout encounters another capillary, with which it connects, allowing blood to circulate. 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. The first sign of tube formation in culture is the appearance in a cell of an elongated vacuole that is at first completely encompassed by cytoplasm (Figure 22-14A). Contiguous cells develop similar vacuoles, and eventually the cells arrange their vacuoles end to end so that the vacuoles become continuous from cell to cell, forming a capillary channel (Figure 22-14B). The process is strongly dependent on the nature of the extracellular matrix in the environment of the cells: formation of capillary tubes is promoted by basal lamina components, such as laminin, which the endothelial cells themselves can secrete. The capillary tubes that develop in a pure culture of endothelial cells do not contain blood, and nothing travels through them, indicating that blood flow and pressure are not required for the formation of a capillary network.

Angiogenesis Is Controlled by Growth Factors Released by the Surrounding Tissues ¹⁴

In living animals endothelial cells form new capillaries wherever there is a need for them. It is thought that when cells in tissues are deprived of oxygen, they release angiogenic factors that induce new capillary growth. Probably for this reason, nearly all cells in a vertebrate are located within 50 mm of a capillary. Similarly, after wounding a burst of capillary growth is stimulated in the neighborhood of the damaged tissue (Figure 22-15). Local irritants or infections also cause a proliferation of new capillaries, most of which regress and disappear when the inflammation subsides.

Angiogenesis is also important in tumor growth. The growth of a solid tumor is limited by its blood supply: if it were not invaded by capillaries, a tumor would be dependent on the diffusion of nutrients from its surroundings and could not enlarge beyond a diameter of a few millimeters. To grow further, a tumor must induce the formation of a capillary network that invades the tumor mass. A small sample of such a tumor implanted in the cornea will cause blood vessels to grow quickly toward the implant from the vascular margin of the cornea, and the growth rate of the tumor increases abruptly as soon as the vessels reach it.

In all of these cases the invading endothelial cells must respond to a signal produced by the tissue that requires a blood supply. The response of the endo-thelial cells includes at least four components. First, the cells must breach the basal lamina that surrounds an existing blood vessel; endothelial cells during angiogenesis have been shown to produce proteases, which enable them to digest their way through the basal lamina of the parent capillary or venule. Second, the endothelial cells must move toward the source of the signal. Third, they must proliferate. Fourth, they must form tubes. In certain circumstances some of the components of this complex response can be elicited in the absence of the others. But there are also identified growth factors that can evoke all four components of the angiogenic response together. Foremost among these factors is a protein known as vascular endothelial growth factor(VEGF - a distant relative of platelet-derived growth factor [PDGF]). This acts selectively on endothelial cells to stimulate angiogenesis in many different circumstances, and it seems to be the agent by which some tumors acquire their rich blood supply. Other growth factors, including some members of the fibroblast growth factor family, also stimulate angiogenesis but at the same time influence other cell types besides endothelial cells. Angiogenic factors such as these are released during tissue repair, inflammation, and tissue growth; they are made by various cell types, including macrophages, mast cells, and fat cells. A number of natural inhibitors have also been identified that can block the formation of new blood vessels. Thus angiogenesis, like the control of cell proliferation in general, seems to be regulated by complex combinations of signals rather than by one signal alone.

Summary

Most populations of differentiated cells in vertebrates are subject to turnover through cell death and cell division. In some cases, such as that of hepatocytes in the liver, the fully differentiated cells simply divide to produce daughter cells of the same type. Both the proliferation and the survival of hepatocytes are controlled to maintain appropriate total cell numbers. If a large part of the liver is destroyed, the remaining hepatocytes increase their division rate to restore the loss; and if hepatocyte proliferation is transiently increased by drug treatment, the increase in cell numbers is soon compensated for by an increase in cell death, returning cell numbers to normal. Such control mechanisms normally keep the numbers of cells of each type in a tissue in appropriate balance. In response to unusual damage, however, repair may be unbalanced, as when the fibroblasts in a repeatedly damaged liver grow too rapidly in relation to the hepatocytes and replace them with connective tissue.

Endothelial cells form a single cell layer that lines all blood vessels and regulates exchanges between the bloodstream and the surrounding tissues. New blood vessels 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. In the living animal anoxic, damaged, or growing tissues stimulate angiogenesis by releasing angiogenic growth factors. These factors attract nearby endothelial cells and stimulate them to secrete proteases, to proliferate, and to form new capillaries.

Introduction

We turn now from cell populations that are renewed by simple duplication to those that are renewed by means of stem cells. These populations vary widely - not only in cell character and rate of turnover, but also in the geometry of cell replacement. In the lining of the small intestine, for example, cells are arranged as a single-layered epithelium. This epithelium covers the surfaces of the villi that project into the lumen of the gut, and it lines the crypts that descend into the underlying connective tissue (Figure 22-16). The stem cells lie in a protected position in the depths of the crypts. The differentiated cells generated from them are carried upward by a sliding movement in the plane of the epithelial sheet until they reach the exposed surfaces of the villi; at the tips of the villi the cells die and are shed into the lumen of the gut. A contrasting example is found in the epithelium that forms the outer covering of the skin, called the epidermis. The epidermis is a many-layered epithelium, and the differentiating cells travel outward from their site of origin in a direction perpendicular to the plane of the cell sheet. In the case of blood cells the spatial pattern of production is complex and appears chaotic. Before going into such details, however, we must pause to consider what a stem cell is.

Stem Cells Can Divide Without Limit and Give Rise to Differentiated Progeny ¹⁶

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 leading irreversibly to terminal differentiation (Figure 22-17).

Stem cells are required wherever there is a recurring need to replace differentiated cells that cannot themselves divide. In several tissues the terminal state of cell differentiation is obviously incompatible with cell division. The cell nucleus may be digested, for example, as in the outermost layers of the skin, or it may be extruded, as in the mammalian red blood cell. Alternatively, the cytoplasm may be heavily encumbered with structures, such as the myofibrils of striated muscle cells, that would hinder mitosis and cytokinesis. In other terminally differentiated cells the chemistry of differentiation may be incompatible with cell division in some more subtle way. In any such case, renewal must depend on stem cells.

The job of the stem cell is not to carry out the differentiated function but rather to produce cells that will. Consequently, stem cells often have a non-descript appearance, making them hard to identify. But that is not to say that stem cells are all alike. Although not terminally differentiated, they are nevertheless determined (see p. 1060): the muscle satellite cell, as a source of skeletal muscle; the epidermal stem cell, as a source of keratinized epidermal cells; the spermatogonium, as a source of spermatozoa; the basal cell of olfactory epi-thelium, as a source of olfactory neurons (Figure 22-18); and so on. Those stem cells that give rise to only one type of differentiated cell are called unipotent, and those that give rise to several cell types are called pluripotent.

Tissues that form from stem cells raise many important questions. We need to consider what factors determine whether a stem cell divides or stays quiescent, what decides whether a given daughter cell remains a stem cell or differentiates, and in what ways the differentiation of a daughter cell is regulated after it has become committed to differentiate. On the opposite side of the balance sheet, we have to consider how cells die and are disposed of and how their survival is controlled. We begin our discussion with the epidermis, for its simple spatial organization makes it relatively easy to study the natural history of its stem cells and the fate of their progeny.

Epidermal Stem Cells Lie in the Basal Layer ^{17, 18}

The epidermal layer of the skin and the epithelial lining of the digestive tract are the two tissues that suffer the most direct and damaging encounters with the external world. In both, mature differentiated cells are rapidly lost from the most exposed positions and are replaced by the proliferation of less differentiated cells in more sheltered niches.

The epidermis is a multilayered epithelium composed largely of keratinocytes (so called because their characteristic differentiated activity is the synthesis of intermediate filament proteins called keratins) (Figure 22-19). 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 normally only these that undergo mitosis. Above the basal cells are several layers of larger prickle cells (Figure 22-20), 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 granular cell layer (see Figure 22-19). This marks the boundary between the inner, metabolically active strata and the outermost layer, 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 containing an intracellular 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 makes them swell slightly, and with suitable staining a remarkably ordered geometric arrangement can often be seen in regions where the skin is thin. The squames are found to be stacked in hexagonal columns that interlock neatly at their edges (Figure 22-21), a typical column being 10-20 cells high and resting on about 10 basal cells.

Differentiating Epidermal Cells Synthesize a Sequence of Different Keratins as They Mature ¹⁸

Having described the static picture, let us now set it in motion. 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 nuclei and cytoplasmic organelles and 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 varies from 2 to 4 weeks, 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 splicing of the gene transcripts. As the new keratinocyte at the base of the column is transformed into the squame at the top (see Figure 22-21), it expresses a succession of different selections from its keratin gene repertoire. During this process other characteristic proteins, such as involucrin, also begin to be synthesized as part of a coordinated program of terminal cell differentiation.

Epidermal Stem Cells Are a Subset of Basal Cells ¹⁹

If each patch of epidermis is maintained indefinitely by proliferation of its basal cells, there must be among these basal cells at least one whose line of descendants will not die out in the lifetime of the animal. We shall call such a cell an immortal stem cell (Figure 22-22). In principle, the division of an immortal stem cell could generate two initially similar daughters whose different fates would be governed by subsequent circumstances. At the opposite extreme, the stem cell division could be always asymmetric: one and only one of the daughters would inherit a special character required for immortality, while the other would be somewhat altered already at the time of its birth in a way that forced it to differentiate and ultimately to die. In the latter case there could never be any increase in the existing number of immortal stem cells, and this is contradicted by the facts. If a patch of epidermis is destroyed, the damage is repaired by surrounding healthy epidermal cells that migrate and proliferate to cover the denuded area. In this process a new self-renewing patch of epidermis is established, implying that additional immortal stem cells have been generated to make up for the loss.

Thus the fate of the daughters of a stem cell must be governed at least partly by the circumstances. One possible determining factor might be contact with the basal lamina or with the exposed connective tissue at a wound, with a loss of contact triggering the start of terminal differentiation, and maintenance of contact tending to preserve stem cell potential. Studies in vitro indicate that this is not the only determinant of basal cell fate, however.

Basal keratinocytes can be dissociated from intact epidermis and will 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 appear 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 appear unable to divide at all, others go through only a few division cycles and then halt, and still others can divide enough times to form large colonies. The basal cells differ also in their expression of extracellular matrix receptors of the integrin family (discussed in Chapter 19): the cells that have more of these receptors, and so are better able to bind to basal laminal components, are the ones with the greater proliferative potential. This suggests that not all basal cells are alike in vivo and that mere contact with the basal lamina is not enough to keep them as stem cells. Rather, it appears that stem cells are a small subset - about 10%of the basal cell population and are programmed to generate a certain proportion of progeny that become committed to terminal differentiation even before they have left the basal layer. In fact, if the keratinocytes are cultured in a Ca²⁺-deficient medium, which keeps them as a monolayer and therefore all in a basal position, some of them will actually embark on terminal differentiation despite their location, as indicated by the synthesis of involucrin; these differentiating cells emerge from the basal layer as soon as the Ca²⁺ concentration is raised.

Nevertheless, contact with extracellular matrix has a critical influence on the choice of cell fate, which is evidently not programmed rigidly. If the cells 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. Some of the cells will refrain from differentiating even in suspension, however, if the medium includes fibronectin (a minor component of basal lamina and a major component of the extracellular matrix that keratinocytes migrate onto during wound healing). The cells that show this response to fibronectin are those that possess appropriate integrins. In normal conditions possession of such receptors presumably holds the cells bound to the basal lamina, keeping open their option to remain as stem cells; loss or inactivation of the receptors leads to ejection from the basal layer, confirming the decision to differentiate; and ejection from the basal layer through other causes leads to loss of the receptors, forcing the cell to differentiate prematurely.

Basal Cell Proliferation Is Regulated According to the Thickness of the Epidermis ²⁰

Whatever the influence of the basal lamina may be, additional controls must operate to regulate the rate of production and the rate of sloughing of epidermal cells. If the outer layers of the epidermis are stripped away, for example, the division rate of the basal cells increases. After a transient overshoot, normal thickness is restored, and the division rate in the basal layer declines to normal. It is as though the removal of the outer differentiated layers releases the cells in the proliferative basal layer from an inhibitory influence, which is restored as soon as the outer layers regain their full thickness.

Although keratinocytes in culture are known to respond to a variety of hormones and growth factors, including epidermal growth factor (EGF), the molecular mechanisms that regulate their proliferation in the body remain an unsolved problem of great clinical importance. The consequences of faulty control of basal cell proliferation are seen in psoriasis. In this common skin disorder 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.

Secretory Cells in the Epidermis Are Secluded in Glands That Have Their Own Population Kinetics ²¹

In certain 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 but have patterns of renewal quite different from those of keratinizing regions.

The mammary gland is of special interest because of the hormonal control of its cell division and differentiation. Milk production must be switched on when a baby is born and switched off when the baby is weaned. A "resting" mammary gland consists of branching systems of ducts embedded in connective tissue; these ducts are lined, in their secretory portions, by a single layer of relatively inactive epithelial cells that serve as stem cells. As a first step toward large-scale milk production, the hormones that circulate during pregnancy cause the duct cells to proliferate and the terminal portions of the ducts to grow and branch, forming little dilated outpocketings, or alveoli, containing secretory cells (Figure 22-23). Milk secretion begins only when these cells are stimulated by the different combination of hormones circulating in the mother after the birth of the baby. Later, when suckling stops, the secretory cells die and most of the alveoli disappear; macrophages rapidly clear away the dead cells, and the gland reverts to its resting state. Degradation of the basal lamina seems to play a critical part in this process of involution.

Cell division in the 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. Mutations in genes involved in these local controls promote the development of cancer, as we discuss in Chapter 24, and it is through studies of breast cancer that several of these control mechanisms have come to light.

Summary

Many tissues, especially those with a rapid turnover - such as the lining of the gut, the epidermal layer of the skin, and the blood-forming tissues - are renewed by means of stem cells. 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. In the skin the stem cells of the epidermis lie in the basal layer, attached to the basal lamina. The progeny of the stem cells differentiate on leaving this layer and, as they move outward, synthesize a succession of different types of keratin until, eventually, their nuclei degenerate, producing an outer layer of dead keratinized cells that are continually shed from the surface. Only a minority of basal cells are stem cells. The fate of the daughters of a stem cell is controlled in part by interactions with the basal lamina and in part by other poorly understood factors. These factors allow two stem cells to be generated from one during repair processes, and they regulate the rate of basal cell proliferation according to the thickness of the epidermis. Glands connected to the epidermis, such as the mammary glands, have their own stem cells and their own distinct patterns of cell renewal.

Introduction

The blood contains many types of cells with very different functions, ranging from the transport of oxygen to the production of antibodies. Some of these cells function entirely within the vascular system, while others use the vascular system only as a means of transport and perform their function elsewhere. All blood cells, however, have certain similarities in their life history. They all have limited life-spans and are produced throughout the life of the animal. Most remarkably, they are all generated ultimately from a common stem cell in the bone marrow. This hemopoietic (or blood-forming) stem cellis thus pluripotent, giving rise to all of the types of terminally differentiated blood cells as well as some other types of cells, such as bone osteoclasts, which we discuss later.

Blood cells can be classified as red or white (Figure 22-24). The red blood cells, or erythrocytes, remain within the blood vessels and transport O₂ and CO₂ bound to hemoglobin. The white blood cells, or leucocytes, combat infection and in some cases phagocytose and digest debris. Leucocytes, unlike erythrocytes, must make their way across the walls of small blood vessels and migrate into tissues to perform their tasks. In addition, the blood contains large numbers of platelets, which are not entire cells but small detached cell fragments or "minicells" derived from the cortical cytoplasm of large cells called megakaryocytes. Platelets adhere specifically to the endothelial cell lining of damaged blood vessels, where they help repair breaches and aid in the process of blood clotting.

There Are Three Main Categories of White Blood Cells: Granulocytes, Monocytes, and Lymphocytes ^{22, 23}

All red blood cells are similar to one another, as are all platelets, but there are many distinct types of white blood cells. They are traditionally grouped into three major categories, called granulocytes, monocytes, and lymphocytes, on the basis of their appearance in the light microscope.

The granulocytes all contain numerous lysosomes and secretory vesicles (or granules) and are subdivided into three classes on the basis of the morphology and staining properties of these organelles (Figure 22-25). 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 small organisms - especially bacteria. Basophilssecrete 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. Eosinophilshelp destroy parasites and modulate allergic inflammatory responses.

Once they leave the bloodstream, monocytes (see Figure 22-25D) 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 removing senescent, dead, and damaged cells in many tissues, and they are unique in being able to ingest large microorganisms such as protozoa.

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 some virus-infected cells. The production of lymphocytes is a specialized topic that is discussed in detail in Chapter 23. 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 ^{22, 24}

Most white blood cells function in tissues other than the blood. The 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 signaling molecules produced locally by mast cells, nerve endings, platelets, and white blood cells, as well as by the activation of complement (discussed in Chapter 23). Some of these signaling 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 selectins (discussed in Chapter 10), and the stronger binding required for the white blood cells to crawl out of the blood vessel is mediated by integrins (discussed in Chapter 19). Other molecules 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 22-26).

Other signaling 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, if one goes to live at high altitude, where oxygen is scarce. Thus blood cell formation (hemopoiesis) necessarily involves complex controls in 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 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. On the other hand, the hemopoietic cells have a nomadic life-style that makes them more accessible to experimental study in other ways. Dispersed hemopoietic cells can be easily transferred, without damage, from one animal to another, and the proliferation and differentiation of individual cells and their progeny can be observed and analyzed in culture. 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 ^{22, 25}

The different types of blood cells and their immediate precursors can be recognized in the bone marrow by their distinctive appearances (Figure 22-27). They are intermingled with one another, as well as with fat cells and other stromal cells (connective-tissue cells) that 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 mm), 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 22-28).

Because of the complex arrangement of the cells in bone marrow, it is difficult to identify 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 there is no visible feature by which the ultimate stem cells can be recognized. To identify and characterize the stem cells, one needs a functional test, 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.

If an animal is exposed to a large dose of x-irradiation, 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 small numbers (about 1 cell in 10,000) that can colonize the irradiated host and permanently reequip it with hemopoietic tissue. One of the tissues where colonies develop is the spleen, which in a normal mouse is an important additional site of hemopoiesis. When the spleen of an irradiated mouse is examined a week or two after the transfusion of cells from a healthy donor, a number of distinct nodules are seen in it, each of which is found to contain a colony of myeloid cells (Figure 22-29); after 2 weeks some colonies may contain more than a million cells. The discreteness of the nodules suggests that each might be a clone of cells descended from a single founder cell, like a bacterial colony on a culture plate; and with the help of genetic markers, it can be established that this is indeed the case.

The founder of such a colony is called a colony-forming cell, or CFC (also known as a colony-forming unit, CFU). The colony-forming cells are heterogeneous. Some give rise to only one type of myeloid cell, while others give rise to mixtures. Some go through many division cycles and form large colonies, while others divide less and form small colonies. Most of the colonies die out after generating a restricted number of terminally differentiated blood cells. A few of the colonies, however, are capable of extensive self-renewal and produce new colony-forming cells in addition to terminally differentiated blood cells. The founders of such self-renewing colonies are assumed to be the hemopoietic stem cells in the transfused bone marrow.

A Pluripotent Stem Cell Gives Rise to All Classes of Blood Cells ²⁶

All the types of myeloid cells can often be found together in one spleen colony, derived from a single stem cell. The hemopoietic stem cell, therefore, is pluri-potent: it can give rise to many cell types. Although the spleen colonies do not seem to contain lymphocytes, another approach shows that these cells also derive from the same stem cell that gives rise to all of the myeloid cells. The demonstration employs genetic markers that make it possible to identify the members of a clone even after they have been released into the bloodstream. Although several types of clonal markers 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, therefore, is 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 not only confirm that all classes of blood cells - both myeloid and lymphoid - derive from a common stem cell (Figure 22-30), but they also make it possible to follow the pedigrees of the blood cells over long periods of time. After many months, when the hemopoietic system has had time to stabilize fully following the transfusion, practically all of the blood cells in the irradiated host mouse are found to be descendants of a remarkably small number - sometimes as few as a single one - of the original transfected cells. A single pluripotent stem cell evidently has the capacity to generate an indefinitely large clone of progeny, among them, presumably, many daughter stem cells with a similar capacity, as well as cells that are terminally differentiated.

The Number of Specialized Blood Cells Is Amplified by Divisions of Committed Progenitor Cells ^{22, 27}

Once a cell has differentiated as an erythrocyte or a granulocyte or some other type of blood cell, there seems to be no going back: the state of differentiation is not reversible. Therefore, at some stage in their development, some of the progeny of the pluripotent stem cell must become irreversibly committed or determined for a particular line of differentiation. It is clear from simple microscopic examination of the bone marrow that this commitment occurs well before the final division in which the mature differentiated cell is formed: one can recognize specialized precursor cells that are still proliferating but already show signs of having begun differentiation. It thus appears that commitment to a particular line of differentiation is followed by a series of cell divisions that amplify the number of cells of a given specialized type.

The hemopoietic system, therefore, can be viewed as a hierarchy of cells. Pluripotent stem cells give rise to committed progenitor cells, which are irreversibly determined as ancestors of only one or a few blood cell types. The committed progenitors divide rapidly but only a limited number of times. At the end of this series of amplification divisions, they develop into terminally differentiated cells, which usually divide no further and die after several days or weeks. Cells may also die at any of the earlier steps in the pathway. Studies in culture provide a way to find out how these cellular events - proliferation, differentiation, and death - are regulated.

The Factors That Regulate Hemopoiesis Can Be Analyzed in Culture ²⁸

Hemopoietic cells will survive, proliferate, and differentiate in culture if, and only if, they are provided with specific growth factors or accompanied by cells that produce these factors; if deprived of such factors, the cells die. Long-term proliferation of pluripotent stem cells can be achieved, for example, by culturing dispersed bone-marrow hemopoietic cells on top of a layer of bone-marrow stromal cells, presumably mimicking the environment in intact bone marrow; such cultures can generate all the types of myeloid cells. Alternatively, 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 be seen to give rise to a clone of thousands of neutrophils. Such culture systems, developed in the mid-1960s, provide 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, or 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 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 kidney and regulates erythropoiesis (the formation of red blood cells).

Erythropoiesis Depends on the Hormone Erythropoietin ²⁹

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 22-31). 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.

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 or 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, 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 programmed cell death.

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. The BFC-E is distinct from the pluripotent 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 the 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). The cell also differs in size from the CFC-E and can be separated from it by sedimentation. Thus the BFC-E is thought to be a progenitor cell committed to erythrocyte differentiation and an early ancestor of the CFC-E (Figure 22-32).

Multiple CSFs Influence the Production of Neutrophils and Macrophages ^{28, 30}

The two professional phagocytic cells, neutrophils and macrophages, develop from a common progenitor cell called the granulocyte/macrophage (or 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 and then die and are phagocytosed by macrophages. Macrophages, by 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 pluripotent 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 (~10^-12 M) by binding to specific cell-surface receptors, as discussed in Chapter 15. A few of these receptors are transmembrane tyrosine kinases. The others belong to another large receptor family (sometimes called the cytokine receptor family), whose members are usually composed of two or more subunits, one of which is frequently shared among several receptor types (Figure 22-33). 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 factors because they are made using recombinant DNA technology) are strong stimulators of hemopoiesis in experimental animals. They are now being 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.

Factors that promote the development of the other classes of myeloid cells, such as megakaryocytes and eosinophils, have also been identified. Again, there are many of these factors, and they have overlapping actions when tested in laboratory assay systems. It is not easy to discover precisely what their individual roles are in natural circumstances. Perhaps the most direct test of the normal function of a CSF is to inactivate the CSF or its receptor in a living animal and study the consequences. This has now been done for several CSFs. Anti-G-CSF antibodies, which neutralize the activity of G-CSF - a CSF that promotes neutrophil production in vitro - have been shown to cause a marked decrease in neutrophils when injected into healthy dogs, establishing that G-CSF is required for the normal production of neutrophils. Genetic approaches can be even more powerful. Mice with a mutation in the gene that encodes M-CSF, for example, are deficient in macrophages, as well as in osteoclasts, which also develop from monocytes. Because osteoclasts are required for bone resorption (as we discuss later), these mice produce an excessive amount of bone, which encroaches on the bone marrow and produces abnormally thickened bones and decreased blood cell formation - a condition called osteopetrosis.

Hemopoietic Stem Cells Depend on Contact with Cells Expressing the Steel Factor ³¹

CSFs that act on the pluripotent stem cells are the most intriguing of all. IL-3, as we have seen, seems to be in this class. Another such factor of fundamental importance came to light through the analysis of mouse mutants that show 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). 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. 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 (Steel) by their environment if progeny cells are to be produced in normal numbers.

Like IL-3, the Steel factor acts on several of the committed blood-cell lineages, including the erythroid lineage, as well as on the pluripotent stem cells. But it has little effect on its own. It mainly potentiates the effects of other CSFs, greatly increasing the number and size of clonal blood-cell colonies of all kinds in culture. It is an unusual CSF in another way too. It is made in both a membrane-bound and a secreted form, generated by alternative splicing of the mRNA, and it seems to be the membrane-bound form that is most important: mutant mice that make the secreted form of the Steel factor but not the membrane-bound form show severe defects. This implies that normal hemopoiesis requires direct cell-cell contact between the hemopoietic stem cell and a stromal cell that expresses Steel and that only this contact enables the Steel factor to activate the Kit receptor protein efficiently. Kit may thus behave as a coreceptor (discussed in Chapter 23), which has to be activated at the same time as receptors for factors such as IL-3 in order to stimulate hemopoiesis. This could help explain why hemopoiesis occurs only in a few special environments, such as that provided by the stromal cells of the bone marrow, while other tissues escape invasion and colonization even though there are always some hemopoietic stem cells circulating in the bloodstream.

The Behavior of a Hemopoietic Cell Depends Partly on Chance ^{28, 32}

Up to this point we have glossed over a central question. The CSFs are defined as factors that promote the production of colonies of differentiated blood cells. But what effect precisely 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 goes through 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 22-34). 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 different effects. Nevertheless, it is still not clear which actions are most important in vivo. The behavior of the pluripotent stem cells remains especially elusive: these crucial cells are few and far between - less than 1 in 1000 of the cells in the bone marrow - and are difficult to identify unambiguously.

Studies in vitro indicate, moreover, that there is a large element of chance in the way a hemopoietic cell behaves. 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 will 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 ³³

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 suicide. 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 the normal regulation of the numbers of blood cells and, as discussed earlier for hepatocytes, of many other cell types too. In many tissues, it seems, cells are programmed to kill themselves if they do not receive specific signals for survival. We have already discussed the importance and the mechanism of programmed cell death during development (see p. 1076); it is no less important in the turnover and renewal of cell populations in the adult body (Figure 22-35). The genes that regulate it have been highly conserved in evolution, to the extent that at least one of them, called bcl-2, coding for an intracellular inhibitor of the cell death program in mammalian cells, can perform the same function in cells of a nematode worm. Too little cell death can be as dangerous to the health of the multicellular organism as too much proliferation, and mutations that inhibit cell death by causing overexpression of bcl-2 have been implicated in the development of cancer, as discussed in Chapter 24.

The amount of programmed cell death in the vertebrate hemopoietic system is enormous: billions of neutrophils die in this way each day in an adult human, for example. Although the mechanism of programmed cell death remains a mystery, the dying cells usually undergo a characteristic morphological change called apoptosis, in which the cell and its nucleus shrink and condense and frequently fragment. By contrast, cells that die accidentally, as a result of acute injury, usually swell and burst - a process called cell necrosis. Whereas cells that die by necrosis spill their cytosolic contents into the extracellular space and elicit an inflammatory response, cells that die by apoptosis disappear in a way that is more efficient for the organism: they are so rapidly phagocytosed by macrophages (or other neighboring cells) that there is no leakage of cytosolic components and no inflammatory response. Once inside the macrophage, the apoptotic cell is quickly disassembled and its chemical building blocks reused.

To activate this disposal mechanism, apoptotic cells change their surface chemistry so that macrophages can recognize them. The recognition mechanism varies depending on the tissue and the type of blood cell. In some cases a lectin on the macrophage surface seems to recognize altered sugar groups on the apoptotic cell surface. In others an integrin (discussed in Chapter 19) on the macrophage surface recognizes an extracellular matrix protein called thrombospondin,which is secreted by the macrophage and seems to act as a bridge between it and the apoptotic cell; the mechanism by which thrombospondin binds to apoptotic cells is unknown. In still other cases the macrophage is thought to recognize phosphatidylserine, a negatively charged phospholipid that is normally confined to the cytosolic leaflet of the plasma membrane lipid bilayer (see Figure 10-11) but apparently relocates to the extracellular leaflet in some apoptotic blood cells. No matter which of these recognition systems is used, macrophages react to the apoptotic cells in a specific way: they engulf and digest them, but they do not secrete inflammation-inducing signals as they do when they phagocytose and digest necrotic cells. This is a second reason why cell necrosis is associated with inflammation, whereas apoptosis is not.

Although biologists have paid much more attention to the control of cell proliferation than to the control of cell survival, it is becoming increasingly clear that both kinds of controls can serve to regulate cell numbers. Both depend on specific signals produced by other cells, ensuring that a cell divides only when more cells are required and that a cell survives only when and where it is needed. The challenge is to define all of the signals that regulate the survival and proliferation of each cell type, to determine how their levels are controlled to balance cell proliferation and cell death according to the varying needs of the organism, and to understand how an individual cell integrates these diverse extracellular signals and decides whether to live or die and whether to divide or remain quiescent.

Summary

The many types of blood cells all derive from a common pluripotent stem cell. In the adult the stem cells are found mainly in bone marrow, where they normally divide infrequently to produce more stem cells (self-renewal) and various committed progenitor cells, each able to give rise to only one or a few types of blood cells. The committed progenitor cells divide profusely under the influence of various protein signaling molecules (called colony-stimulating factors, or CSFs) and then differentiate into mature blood cells, which usually die after several days or weeks. Studies of hemopoiesis have been greatly aided byin vitro assays in which stem cells or committed progenitor cells form clonal colonies when cultured in a semisolid matrix. The progeny of stem cells appear to make their choices among alternative developmental pathways in a partly random manner. Cell death, controlled by the availability of CSFs, also plays a central part in regulating the numbers of mature differentiated blood cells; it depends on activation of an intracellular suicide program and is thought to help regulate cell numbers in many other tissues and in other kinds of animals.

Introduction

The term "muscle" covers a multitude of cell types, all specialized for contraction but in other respects dissimilar. As noted in Chapter 16, a contractile system involving actin and myosin is a basic feature of animal cells in general, but muscle cells have developed this apparatus to a high degree. Mammals possess four main categories of cells specialized for contraction: skeletal muscle cells, heart (or cardiac) muscle cells, smooth muscle cells, and myoepithelial cells (Figure 22-36). These differ in function, structure, and development. Although all of them appear to generate contractile forces by means of organized filament systems based on actin and myosin, the actin and myosin molecules employed are somewhat different in amino acid sequence, are differently arranged in the cell, and are associated with different sets of proteins to control contraction.

Skeletal muscle cells, whose contractile apparatus is discussed in detail in Chapter 16, are responsible for practically all movements that are under voluntary control. These cells can be very large (2 or 3 cm long and 100 mm in diameter in an adult human) and are often referred to as muscle fibers because of their highly elongated shape. Each one is a syncytium, containing many nuclei within a common cytoplasm. The other types of muscle cells are more conventional, having only a single nucleus. Heart muscle cells resemble skeletal muscle cells in that their actin and myosin filaments are aligned in very orderly arrays to form a series of contractile units called sarcomeres, so that the cells have a striated appearance. Smooth muscle cells are so called because they, in contrast, do not appear striated. The functions of smooth muscle vary greatly, from propelling food along the digestive tract to erecting hairs in response to cold or fear. Myoepithelial cells also have no striations, but unlike all other muscle cells they lie in epithelia and are derived from the ectoderm. They form the dilator muscle of the iris and serve to expel saliva, sweat, and milk from the corresponding glands (see Figure 22-36E). The four main categories of muscle cells can be further divided into distinctive subtypes, each with its own characteristic features.

The mechanisms of muscle contraction are discussed in Chapter 16; here, we consider how muscle tissue is generated and maintained. We focus on the skeletal muscle cell, which has a curious mode of development, a striking ability to modulate its differentiated character, and an unusual strategy for repair.

New Skeletal Muscle Cells Form by the Fusion of Myoblasts ^{2, 35}

The previous chapter described how certain cells, originating from the somites of a vertebrate embryo at a very early stage, become determined as myoblasts (that is, as precursors of skeletal muscle cells) and migrate into the adjacent embryonic connective tissue, or mesenchyme. As discussed in Chapter 9, the commitment to be a myoblast (rather than, say, a fibroblast) depends on the activation of one or more myogenic genes, which encode gene regulatory proteins of the helix-loop-helix family. After a period of proliferation the myoblasts fuse with one another to form multinucleate skeletal muscle cells (Figure 22-37). As they fuse, they undergo a dramatic switch of phenotype that depends on the coordinated activation of a whole battery of muscle-specific genes. Once fusion has occurred, the nuclei never again replicate their DNA. Fusion involves specific cell-cell adhesion molecules that mediate recognition between myoblasts.

Myoblasts that have been kept proliferating in culture for as long as 2 years still retain the ability to differentiate and will fuse to form muscle cells in response to a suitable change in culture conditions. Fibroblast growth factor (FGF) in the medium seems to be crucial for keeping the myoblasts proliferating and preventing their differentiation: if FGF is removed, the cells rapidly stop dividing, fuse, and differentiate. The system of controls is complex, however, and in order to differentiate, myoblasts must attach to the extracellular matrix. Moreover, the process of fusion is cooperative: fusing myoblasts secrete factors that encourage other myoblasts to fuse. 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 That They Contain ³⁶

Once formed, a skeletal muscle cell is generally retained for the entire lifetime of the animal. Over this period it 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 cell matures, different selections of 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 cells, each with different sets of protein isoforms and different functional properties, can be found side by side (Figure 22-38).

Some Myoblasts Persist as Quiescent Stem Cells in the Adult ³⁷

A muscle can grow in three ways: its differentiated muscle cells can increase in number, in length, or in girth. Because skeletal muscle cells are unable to divide, more of them can be made only by the fusion of myoblasts. The adult number of multinucleated skeletal muscle cells is in fact attained early - before birth in humans. The subsequent enormous increase in muscle bulk is achieved by cell enlargement. Growth in length depends on recruitment of more myoblasts into the existing multinucleate cells, mainly by fusion at their ends, which increases the number of nuclei in each cell. In contrast, growth in girth, such as occurs in the muscles of weightlifters, depends on an increase in the size and numbers of the contractile myofibrils that each muscle cell contains rather than on changes in the numbers of muscle cells or of their nuclei.

In the adult, nevertheless, a few myoblasts persist as small, flattened, and inactive cells lying in close contact with the mature muscle cell and contained within its sheath of basal lamina. If the muscle is damaged or if it is treated artificially with FGF, these so-called satellite cells are activated to proliferate (Figure 22-39), and their progeny can fuse to form new muscle cells. 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.

Although this muscle repair mechanism operates well in small animals such as mice, it is less efficient in humans. In muscular dystrophy, for example, differentiated skeletal muscle cells die because of a genetic defect in the cytoskeletal dystrophin protein (see p. 855). As a result, satellite cells proliferate to form new muscle cells; but this regenerative response is unable to keep pace with the damage, and the muscle cells are eventually replaced by connective tissue, blocking any further possibility of regeneration.

Summary

Skeletal muscle cells are one of the four main categories of vertebrate cells specialized for contraction, and they are responsible for voluntary movement. Each skeletal muscle cell is a syncytium and develops by the fusion of many myoblasts. Myoblasts are stimulated to proliferate by growth factors such as FGF, but once they fuse, they can no longer divide. Myoblast fusion is generally coupled with the onset of muscle cell 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.

Introduction

Many of the differentiated cells in the adult body can be grouped into families whose members are closely related by origin and by character. An important example is the family of connective-tissue cells, whose members are not only related but are also to an unusual extent interconvertible. The family includes fibroblasts, cartilage cells, and bone cells, all of which are specialized for secretion of collagenous extracellular matrix and are jointly responsible for the architectural framework of the body, as well as fat cells and smooth muscle cells, which appear to have a common origin with them. These cell types and the interconversions that are thought to occur between them are illustrated in Figure 22-40. Connective-tissue cells play a central part in the support and repair of almost every tissue and organ, and the adaptability of their differentiated character is an important feature of the responses to many types of damage.

Fibroblasts Change Their Character in Response to Signals in the Extracellular Matrix ^{38, 39}

Fibroblasts appear 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 migrate into the wound, proliferate, 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 life-style, 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 22-41).

As indicated in Figure 22-40, 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 some important uncertainties about their interconversions, however. There is good evidence that fibroblasts in different parts of the body are intrinsically different, and it is far from proven that all fibroblasts in a given region are equivalent. In the absence of firm evidence to the contrary, it is simplest to suppose that they are indeed equivalent, but it is conceivable that connective tissue may contain a mixture of distinct fibroblast lineages, some capable of transformation into chondrocytes, others capable of transformation into fat cells, and so on, rather than just one type of fibroblast with multiple developmental capabilities. It is possible also that "mature" fibroblasts incapable of transformation may exist side by side with "immature" fibroblasts (often called mesenchymal cells) that can develop into a variety of mature cell types.

Despite these uncertainties, there is clear evidence, from studies both in vivo and in vitro, that connective-tissue cells can undergo radical changes of character. Thus, 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 dermal fibroblasts) become transformed into cartilage cells and, a little later, others into bone cells, thereby creating a small lump of bone, complete with a marrow cavity. These experiments suggest that components in the extracellular matrix can dramatically influence connective-tissue cell differentiation. We shall see that similar cell transformations are important in the natural repair of broken bones. In fact, bone matrix has been found to contain trapped within it high concentrations of several growth factors that can affect the behavior of connective-tissue cells, including, in particular, transforming growth factor β (TGF-β) and a set of distinct bone morphogenetic proteins (BMPs) that belong to the TGF-β superfamily. 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 formation of cartilage, of bone, or of fibrous matrix, according to the site and circumstances of injection.

The Extracellular Matrix May Influence Connective-Tissue Cell Differentiation by Affecting Cell Shape and Attachment ⁴⁰

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 will 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. However, under conditions where the cells are kept at relatively low density and remain as a monolayer on the culture dish, a transformation occurs. The cells lose the rounded shape that is typical of chondrocytes, flatten down on the substratum, and stop making cartilage matrix. In particular, they stop producing type II collagen - the type characteristic of cartilage - and instead start producing type I collagen - the type 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 of cell shape and attachments. 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, as we saw in Chapter 16.

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. 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.

Different Signaling Molecules Act Sequentially to Regulate Production of Fat Cells ⁴¹

Fat cells, or adipocytes, are also thought to develop from fibroblastlike cells, both during normal mammalian development and in various pathological circumstances - for example, in muscular dystrophy, where the muscle cells die and are gradually replaced by fatty connective tissue. Fat cell differentiation begins with the production of specific enzymes, followed by the accumulation of fat droplets, which then coalesce and enlarge until the cell is hugely distended, with only a thin rim of cytoplasm around the mass of lipid (Figure 22-42).

The process can be studied in culture (using fibroblast cell lines such as mouse 3T3 cells) so that the factors that influence it can be analyzed. It was initially found that the development of fat cells in culture required the presence of fetal calf serum, a common additive to culture media. The crucial factor in the serum that triggers fat cell differentiation was later identified as growth hormonea protein normally secreted into the bloodstream by the pituitary gland. There is evidence that growth hormone stimulates chondrocyte as well as fat cell differentiation and that it acts in this way in vivo as well as in vitro. But growth hormone is not the only secreted signaling molecule that regulates fat cell development. Fat cell precursors that have been stimulated by growth hormone become sensitive to IGF-1 (insulinlike growth factor-1), which stimulates the proliferation of the differentiating fat cells.

The differentiation of fat cells, like that of chondrocytes, is also influenced by factors that affect cell shape and anchorage. The differentiation of 3T3 cells into fat cells is inhibited if the cells are allowed to flatten onto a culture dish coated with fibronectin, to which they adhere strongly. This inhibition is reversed, however, by treatment with the drug cytochalasin, which disrupts actin filaments and causes the cells to round up.

All of these experiments on connective-tissue cells illustrate a recurrent theme: differentiation is regulated by a combination of soluble signals and contacts with the extracellular matrix. The effects of each of these factors depend on the character of the responding cell, and this in turn depends on the cell's developmental history.

Bone Is Continually Remodeled by the Cells Within It ⁴²

Bone is a very dense, specialized form of connective tissue. 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. Throughout its 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 demolishes old bone matrix while another deposits new bone matrix. This mechanism provides for continuous turnover and replacement of the matrix in the interior of the bone.

Bone can grow only by apposition - that is, by the laying down of additional matrix and cells on the free surfaces of the hard tissue. This process must occur in the embryo 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 the bones are first formed out of cartilage. Each scale model 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.

Osteoblasts Secrete Bone Matrix, While Osteoclasts Erode It ^{40, 42, 43}

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 22-43). 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 22-44). 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 22-45). 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 22-46). To produce the plywoodlike structure of compact bone, these osteoblasts lay down concentric layers of new bone, 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 22-47).

There are many unsolved problems about these processes. Bones, for example, have a remarkable ability to adapt to the load imposed on them by remodeling their structure, and this implies that the deposition and erosion of the matrix are somehow controlled by local mechanical stresses. We do not understand the mechanisms that determine whether matrix will be deposited by osteoblasts or eroded by osteoclasts at a given bone surface, but it seems likely that an important part is played by growth factors that are made by the bone cells, trapped in the matrix, and released, perhaps, when the matrix is degraded or suitably stressed.

During Development, Cartilage Is Eroded by Osteoclasts to Make Way for Bone ⁴⁴

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 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 22-48).

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 will carry out a repair by a rough-and-ready recapitulation of the original embryonic process, in which cartilage is first laid down to bridge the gap and is then replaced by bone.

The Structure of the Body Is Stabilized by Its Connective-Tissue Framework and by the Selective Cohesion of Cells ⁴⁵

A bone, like the body as a whole, is a dynamic system, maintaining its structure through a balance between the opposed activities of a variety of specialized cells. Any dynamic system poses a problem of stability, and this leads us to a general question about the maintenance of body structure. We have seen how cells in various types of tissues maintain their differentiated state, how new cells are produced in a controlled fashion to replace those that are lost, and how the extracellular matrix is remodeled and renewed. But why do the different types of cells not become progressively jumbled and misplaced? Why does the whole structure not sag, warp, or otherwise change its proportions as new parts are substituted for old?

To some extent, of course, the body does sag and warp with the passage of time - that is a part of aging. But it does so remarkably little. The skeleton, despite constant remodeling, provides a rigid framework whose dimensions scarcely change. This is partly because the parts of a bone are renewed not all at once but little by little, rather like a building whose bricks are replaced one at a time. Besides such conservatism in the mode of renewal, active homeostatic mechanisms are at work. Thus small departures of a bone from its normal shape set up altered patterns of stresses, which regulate bone remodeling in such a way as to restore the bone to its normal shape (Figure 22-49).

The growth and renewal of many of the soft parts of the body are also homeostatically controlled so that each component is adjusted to fit its niche. The epidermis spreads to keep the surface of the body covered; if it is damaged, the cells grow back to cover the lesion, halting their migration when that end is achieved; connective tissue grows to just the extent necessary to fill the gap created by a wound; and so on. In all this, something more than mere control of cell numbers is required. The various types of differentiated cells must be maintained not only in the correct relative quantities but also in the correct relative positions. Tissue turnover necessarily involves cell movements. Somehow those movements must be limited; the cells must be subject to territorial restraints.

These restraints are of various kinds. Glands and other masses of specialized cells are often contained within tough capsules of connective tissue. Many types of cells die if they find themselves outside their normal environment, deprived of specific growth factors on which their survival depends. Perhaps the most important strategy for keeping the different cells in their places is the strategy of selective cell-cell adhesion: cells of the same type tend to stick together, either in solid masses, such as smooth muscle, or in epithelial sheets, such as the lining of the gut. As described in Chapter 19, this mechanism, for example, enables dissociated epidermal cells to reassociate spontaneously to form a correctly structured epithelium. And on a larger scale, stable epithelial sheets of cells serve to divide the body into compartments, thereby keeping other cells properly segregated and confined to their correct territories.

Clearly, the checks and balances that preserve the structure of the body and the organization of its cells in the face of continual turnover and renewal are intricate and subtle. The importance of these controls is all too clearly evident when they fail, as we see when we come to the topic of cancer in the final chapter of this book.

Summary

The family of connective-tissue cells includes fibroblasts, cartilage cells, bone cells, fat cells, and smooth muscle cells. Fibroblasts seem to be able to transform into any of the other members of the family - in some cases reversibly - although it is not clear whether this is a property of a single type of fibroblast that is pluripotent or of a mixture of distinct types of fibroblasts with more restricted potentials. 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.

Cartilage and bone both consist of cells embedded in a solid matrix. Cartilage has a deformable matrix and can grow by swelling, whereas bone is rigid and can grow only by accretion at its surfaces. Bone is, nonetheless, subject to perpetual remodeling through 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. Although bone, like most other tissues, is subject to continual turnover, this dynamic process is regulated so that the global structure is preserved. In this way, and through other mechanisms such as selective cell-cell adhesion, the organization of the body is stably maintained even though most of its components are continually being replaced.

Cells of the Adult Human Body: A Catalogue

How many distinct cell types are there in an adult human being? In other words, how many normal adult ways are there of expressing the human genome? A large textbook of histology will mention about 200 cell types that qualify for individual names. These traditional names are not, like the names of colors, labels for parts of a continuum that has been subdivided arbitrarily: they represent, for the most part, discrete and distinctly different categories. Within a given category there is often some variation - the skeletal muscle fibers that move the eyeball are small, while those that move the leg are big; auditory hair cells in different parts of the ear may be tuned to different frequencies of sound; and so on. But there is no continuum of adult cell types intermediate in character between, say, the muscle cell and the auditory hair cell.

The traditional histological classification is based on the shape and structure of the cell as seen in the microscope and on its chemical nature as assessed very crudely from its affinities for various stains. Subtler methods reveal new subdivisions within the traditional classification. Thus modern immunology has shown that the old category of "lymphocyte" includes more than 10 quite distinct cell types. Similarly, pharmacological and physiological tests reveal that there are many varieties of smooth muscle cell - those in the wall of the uterus, for example, are highly sensitive to estrogen, and in the later stages of pregnancy to oxytocin, while those in the wall of the gut are not. Another major type of diversity is revealed by embryological experiments of the sort discussed in Chapter 21. These show that, in many cases, apparently similar cells from different regions of the body are nonequivalent, that is, they are inherently different in their developmental capacities and in their effects on other cells. Thus, within categories such as "fibroblast" there are probably many distinct cell types, different chemically in ways that are not easy to perceive directly.

For these reasons any classification of the cell types in the body must be somewhat arbitrary with respect to the fineness of its subdivisions. Here, we list only the adult human cell types that a histology text would recognize to be different, grouped into families roughly according to function. We have not attempted to subdivide the class of neurons of the central nervous system. Also, where a single cell type such as the keratinocyte is conventionally given a succession of different names as it matures, we give only two entries - one for the differentiating cell and one for the stem cell. With these serious provisos, the 210 varieties of cells in the catalogue represent a more or less exhaustive list of the distinctive ways in which a given mammalian genome can be expressed in the phenotype of a normal cell of the adult body.

Keratinizing Epithelial Cells

• keratinocyte of epidermis (= differentiating epidermal cell)

• basal cell of epidermis (stem cell)

• keratinocyte of fingernails and toenails

• basal cell of nail bed (stem cell)

• hair shaft cells

  • medullary

  • cortical

  • cuticular

• hair-root sheath cells

  • cuticular

  • of Huxley's layer

  • of Henle's layer

  • external

• hair matrix cell (stem cell)

Cells of Wet Stratified Barrier Epithelia

• surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, vagina

• basal cell of these epithelia (stem cell)

• cell of urinary epithelium (lining bladder and urinary ducts)

Epithelial Cells Specialized for Exocrine Secretion

• cells of salivary gland

  • mucous cell (secretion rich in polysaccharide)

  • serous cell (secretion rich in glycoprotein enzymes)

• cell of von Ebner's gland in tongue (secretion to wash over taste buds)

• cell of mammary gland, secreting milk

• cell of lacrimal gland, secreting tears

• cell of ceruminous gland of ear, secreting wax

• cell of eccrine sweat gland, secreting glycoproteins (dark cell)

• cell of eccrine sweat gland, secreting small molecules (clear cell)

• cell of apocrine sweat gland (odoriferous secretion, sex-hormone sensitive)

• cell of gland of Moll in eyelid (specialized sweat gland)

• cell of sebaceous gland, secreting lipid-rich sebum

• cell of Bowman's gland in nose (secretion to wash over olfactory epithelium)

• cell of Brunner's gland in duodenum, secreting alkaline solution of mucus and enzymes

• cell of seminal vesicle, secreting components of seminal fluid, including fructose (as fuel for swimming sperm)

• cell of prostate gland, secreting other components of seminal fluid

• cell of bulbourethral gland, secreting mucus

• cell of Bartholin's gland, secreting vaginal lubricant

• cell of gland of Littré, secreting mucus

• cell of endometrium of uterus, secreting mainly carbohydrates

• isolated goblet cell of respiratory and digestive tracts, secreting mucus

• mucous cell of lining of stomach

• zymogenic cell of gastric gland, secreting pepsinogen

• oxyntic cell of gastric gland, secreting HCl

• acinar cell of pancreas, secreting digestive enzymes and bicarbonate

• Paneth cell of small intestine, secreting lysozyme

• type II pneumocyte of lung, secreting surfactant

• Clara cell of lung (function unknown)

Cells Specialized for Secretion of Hormones

• cells of anterior pituitary, secreting

  • growth hormone

  • follicle-stimulating hormone

  • luteinizing hormone

  • prolactin

  • adrenocorticotropic hormone

  • thyroid-stimulating hormone

• cell of intermediate pituitary, secreting

  • melanocyte-stimulating hormone

• cells of posterior pitutiary, secreting

  • oxytocin

  • vasopressin

• cells of gut and respiratory tract, secreting

  • serotonin

  • endorphin

  • somatostatin

  • gastrin

  • secretin

  • cholecystokinin

  • insulin

  • glucagon

  • bombesin

• cells of thyroid gland, secreting

  • thyroid hormone

  • calcitonin

• cells of parathyroid gland, secreting

  • parathyroid hormone

  • oxyphil cell (function unknown)

• cells of adrenal gland, secreting

  • epinephrine

  • norepinephrine

  • steroid hormones

    • mineralocorticoids

    • glucocorticoids

• cells of gonads, secreting

  • testosterone (Leydig cell of testis)

  • estrogen (theca interna cell of ovarian follicle)

  • progesterone (corpus luteum cell of ruptured ovarian follicle)

• cells of juxtaglomerular apparatus of kidney

  • juxtaglomerular cell (secreting renin)

  • macula densa cell

    • (uncertain but probably related in function; possibly involved secretion of erythropoietin)

  • Peripolar cell

    • (uncertain but probably related in function; possibly involved secretion of erythropoietin)

  • mesangial cel

    • (uncertain but probably related in function; possibly involved secretion of erythropoietin)

Epithelial Absorptive Cells in Gut, Exocrine Glands, and Urogenital Tract

• brush border cell of intestine (with microvilli)

• striated duct cell of exocrine glands

• gall bladder epithelial cell

• brush border cell of proximal tubule of kidney

• distal tubule cell of kidney

• nonciliated cell of ductulus efferens

• epididymal principal cell

• epididymal basal cell

Cells Specialized for Metabolism and Storage

• hepatocyte (liver cell)

• fat cells

  • white fat

  • brown fat

  • lipocyte of liver

Epithelial Cells Serving Primarily a Barrier Function, Lining the Lung, Gut, Exocrine Glands, and Urogenital Tract

• type I pneumocyte (lining air space of lung)

• pancreatic duct cell (centroacinar cell)

• nonstriated duct cell of sweat gland, salivary gland, mammary gland, etc. (various)

• parietal cell of kidney glomerulus

• podocyte of kidney glomerulus

• cell of thin segment of loop of Henle (in kidney)

• collecting duct cell (in kidney)

• duct cell of seminal vesicle, prostate gland, etc. (various)

Epithelial Cells Lining Closed Internal Body Cavities

• vascular endothelial cells of blood vessels and lymphatics

  • fenestrated

  • continuous

  • splenic

• synovial cell (lining joint cavities, secreting largely hyaluronic acid)

• serosal cell (lining peritoneal, pleural, and pericardial cavities)

• squamous cell lining perilymphatic space of ear

• cells lining endolymphatic space of ear

  • squamous cell

  • columnar cells of endolymphatic sac

    • with microvilli

    • without microvilli

  • "dark" cell

  • vestibular membrane cell

  • stria vascularis basal cell

  • stria vascularis marginal cell

  • cell of Claudius

  • cell of Boettcher

• choroid plexus cell (secreting cerebrospinal fluid)

• squamous cell of pia-arachnoid

• cells of ciliary epithelium of eye

  • pigmented

  • nonpigmented

• corneal "endothelial" cell

Ciliated Cells with Propulsive Function

• of respiratory tract

• of oviduct and of endometrium of uterus (in female)

• of rete testis and ductulus efferens (in male)

• of central nervous system (ependymal cell lining brain cavities)

Cells Specialized for Secretion of Extracellular Matrix

• epithelial

  • ameloblast (secreting enamel of tooth)

  • planum semilunatum cell of vestibular apparatus of ear (secreting proteoglycan)

  • interdental cell of organ of Corti (secreting tectorial "membrane" covering hair cells of organ of Corti)

• nonepithelial (connective tissue)

  • fibroblasts (various - of loose connective tissue, of cornea, of tendon, of reticular tissue of bone marrow, etc.)

  • pericyte of blood capillary

  • nucleus pulposus cell of intervertebral disc

  • cementoblast/cementocyte (secreting bonelike cementum of root of tooth)

  • odontoblast/odontocyte (secreting dentin of tooth)

  • chondrocytes

    • of hyaline cartilage

    • of fibrocartilage

    • of elastic cartilage

  • osteoblast/osteocyte

  • osteoprogenitor cell (stem cell of osteoblasts)

  • hyalocyte of vitreous body of eye

  • stellate cell of perilymphatic space of ear

Contractile Cells

• skeletal muscle cells

  • red (slow)

  • white (fast)

  • intermediate

  • muscle spindlenuclear bag

  • muscle spindlenuclear chain

  • satellite cell (stem cell)

• heart muscle cells

  • ordinary

  • nodal

  • Purkinje fiber

• smooth muscle cells (various)

• myoepithelial cells

  • of iris

  • of exocrine glands

Cells of Blood and Immune System

• red blood cell

• megakaryocyte

• macrophages and related cells

  • monocyte

  • connective-tissue macrophage (various)

  • Langerhans cell (in epidermis)

  • osteoclast (in bone)

  • dendritic cell (in lymphoid tissues)

  • microglial cell (in central nervous system)

• neutrophil

• eosinophil

• basophil

• mast cell

• T lymphocyte

  • helper T cell

  • suppressor T cell

  • killer T cell

• B lymphocyte

  • IgM

  • IgG

  • IgA

  • IgE

• killer cell

• stem cells and committed progenitors for the blood and immune system (various)

Sensory Transducers

• photoreceptors

  • rod

  • cones

    • blue sensitive

    • green sensitive

    • red sensitive

• hearing

  • inner hair cell of organ of Corti

  • outer hair cell of organ of Corti

• acceleration and gravity

  • type I hair cell of vestibular apparatus of ear

  • type II hair cell of vestibular apparatus of ear

• taste

  • type II taste bud cell

• smell

  • olfactory neuron

  • basal cell of olfactory epithelium (stem cell for olfactory neurons)

• blood pH

  • carotid body cell

    • type I

    • type II

• touch

  • Merkel cell of epidermis

  • primary sensory neurons specialized for touch (various)

• temperature

  • primary sensory neurons specialized for temperature

    • cold sensitive

    • heat sensitive

• pain

  • primary sensory neurons specialized for pain (various)

• configurations and forces in musculoskeletal system

  • proprioceptive primary sensory neurons (various)

Autonomic Neurons

• cholinergic (various)

• adrenergic (various)

• peptidergic (various)

Supporting Cells of Sense Organs and of Peripheral Neurons

• supporting cells of organ of Corti

  • inner pillar cell

  • outer pillar cell

  • inner phalangeal cell

  • outer phalangeal cell

  • border cell

  • Hensen cell

• supporting cell of vestibular apparatus

• supporting cell of taste bud (type I taste bud cell)

• supporting cell of olfactory epithelium

• Schwann cell

• satellite cell (encapsulating peripheral nerve cell bodies)

• enteric glial cell

Neurons and Glial Cells of Central Nervous System

• neurons (huge variety of types - still poorly classified)

• glial cells

  • astrocyte (various)

  • oligodendrocyte

Lens Cells

• anterior lens epithelial cell

• lens fiber (crystallin-containing cell)

Pigment Cells

• melanocyte

• retinal pigmented epithelial cell

Germ Cells

• oogonium/oocyte

• spermatocyte

• spermatogonium (stem cell for spermatocyte)

Nurse Cells

• ovarian follicle cell

• Sertoli cell (in testis)

• thymus epithelial cell