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Cooper5e_Ch17 - UNCORRECTED PAGE PROOFS 17 Programmed Cell...

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Unformatted text preview: UNCORRECTED PAGE PROOFS 17 Programmed Cell Death 00 Stem Cells and the Maintenance of Adult Tissues 00 Embryonic Stem Cells and Therapeutic Cloning 00 KEY EXPERIMENT Identification of Genes Required for Programmed Cell Death 000 KEY EXPERIMENT Culture of Embryonic Stem Cells 000 Cell Death and Cell Renewal Cell death and cell proliferation are balanced throughout the life of multicellular organisms. Animal development begins with the rapid proliferation of embryonic cells, which then differentiate to produce the many specialized types of cells that make up adult tissues and organs. Whereas the nematode C. elegans consists of only 959 somatic cells, humans possess a total of approximately 1014 cells, consisting of more than 200 differentiated cell types. Starting from only a single cell—the fertilized egg—all the diverse cell types of the body are produced and organized into tissues and organs. This complex process of development involves not only cell proliferation and differentiation but also cell death. Although cells can die as a result of unpredictable traumatic events, such as exposure to toxic chemicals, most cell deaths in multicellular organisms occur by a normal physiological process of programmed cell death, which plays a key role both in embryonic development and in adult tissues. In adult organisms, cell death must be balanced by cell renewal, and most tissues contain stem cells that are able to replace cells that have been lost. Abnormalities of cell death are associated with a wide variety of illnesses, including cancer, autoimmune disease, and neurodegenerative disorders, such as Parkinson’s and Alzheimer’s disease. Conversely, the ability of stem cells to proliferate and differentiate into a wide variety of cell types has generated enormous interest in the possible use of these cells, particularly embryonic stem cells, to replace damaged tissues. The mechanisms and regulation of cell death and cell renewal have therefore become areas of research at the forefront of biology and medicine. Programmed Cell Death Programmed cell death is carefully regulated so that the fate of individual cells meets the needs of the organism as a whole. In adults, programmed cell death is responsible for balancing cell proliferation and maintaining constant cell numbers in tissues undergoing cell turnover. For example, about 5 × 1011 blood cells are eliminated daily in humans by programmed cell death, balancing their continual production in the bone marrow. In addition, programmed cell death provides a defense mechanism by which damaged and potentially dangerous cells can be eliminated for the good of the organism as a whole. Virus-infected cells frequently undergo proThis material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS 2 CHAPTER 17 grammed cell death, thereby preventing the production of new virus particles and limiting spread of the virus through the host organism. Other types of cellular insults, such as DNA damage, also induce programmed cell death. In the case of DNA damage, programmed cell death may eliminate cells carrying potentially harmful mutations, including cells with mutations that might lead to the development of cancer. During development, programmed cell death plays a key role by eliminating unwanted cells from a variety of tissues. For example, programmed cell death is responsible for the elimination of larval tissues during amphibian and insect metamorphosis, as well as for the elimination of tissue between the digits during the formation of fingers and toes. Another wellcharacterized example of programmed cell death is provided by development of the mammalian nervous system. Neurons are produced in excess, and up to 50% of developing neurons are eliminated by programmed cell death. Those that survive are selected for having made the correct connections with their target cells, which secrete growth factors that signal cell survival by blocking the neuronal cell death program. The survival of many other types of cells in animals is similarly dependent on growth factors or contacts with neighboring cells or the extracellular matrix, so programmed cell death is thought to play an important role in regulating the associations between cells in tissues. The Events of Apoptosis In contrast to the accidental death of cells that results from an acute injury (necrosis), programmed cell death is an active process, which usually proceeds by a distinct series of cellular changes known as a poptosis , first described in 1972 (Figure 17.1). During apoptosis, chromosomal DNA is (A) (B) (C) Hours after induction of apoptosis 0 1 2 3 DNA fragmentation Chromatin condensation Normal Fragmentation of nucleus Fragmentation of the cell Apoptotic FIGURE 17.1 Apoptosis (A) Diagrammatic representation of the events of apoptosis. (B) Light micrographs of normal and apoptotic human leukemia cells illustrating chromatin condensation and nuclear fragmentation. (C) Gel electrophoresis of DNA from apoptotic cells, showing its degradation to fragments corresponding to multiples of 200 base pairs (the size of nucleosomes) at 1–4 hours following induction of apoptosis. (B, courtesy of D. R. Green/La Jolla Institute for Allergy and Immunology; C, courtesy of Ken Adams, Boston University.) This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL Normal cell Cytosol Outside of cell – – – Phosphatidylserine FIGURE 17.2 Phagocytosis of apoptotic cells Apoptotic cells and cell fragments are recognized and engulfed by phagocytic cells. One of the signals recognized by phagocytes is phosphatidylserine on the cell surface. In normal cells, phosphatidylserine is restricted to the inner leaflet of the plasma membrane, but it becomes expressed on the cell surface during apoptosis. Apoptosis Receptor – – – Phagocytic cell usually fragmented as a result of cleavage between nucleosomes. The chromatin condenses and the nucleus then breaks up into small pieces. Finally, the cell itself shrinks and breaks up into membrane-enclosed fragments called apoptotic bodies. Apoptotic cells and cell fragments are efficiently recognized and phagocytosed by both macrophages and neighboring cells, so cells that die by apoptosis are rapidly removed from tissues. In contrast, cells that die by necrosis swell and lyse, releasing their contents into the extracellular space and causing inflammation. The removal of apoptotic cells is mediated by the expression of so-called “eat me” signals on the cell surface. These signals include phosphatidylserine, which is normally restricted to the inner leaflet of the plasma membrane (see Figure 13.2). During apoptosis, phosphatidylserine becomes expressed on the cell surface where it is recognized by receptors expressed by phagocytic cells (Figure 17.2). Pioneering studies of programmed cell death during the development of C. elegans provided the critical initial insights that led to understanding the molecular mechanism of apoptosis. These studies in the laboratory of Robert Horvitz initially identified three genes that play key roles in regulating and executing apoptosis. During normal nematode development, 131 somatic cells out of a total of 1090 are eliminated by programmed cell death, yielding the 959 somatic cells in the adult worm. The death of these cells is highly specific, such that the same cells always die in developing embryos. Based on this developmental specificity, Robert Horvitz undertook a genetic analysis of cell death in C. elegans with the goal of identifying the genes responsible for these developmental cell deaths. In 1986 mutagenesis of C. elegans identified two genes, ced-3 and ced-4, that were required for developmental cell death. If either ced-3 or ced-4 was inactivated by mutaThis material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. 3 The term apoptosis is derived from the Greek word describing the falling of leaves from a tree or petals from a flower. It was coined to differentiate this form of programmed cell death from the accidental cell deaths caused by inflammation or injury. 17.1 WEBSITE ANIMATION Apoptosis During apoptosis, chromosomal DNA is usually fragmented, the chromatin condenses, the nucleus breaks up, and the cell shrinks and breaks into apoptotic bodies. UNCORRECTED PAGE PROOFS 4 CHAPTER 17 KEY EXPERIMENT Identification of Genes Required for Programmed Cell Death Genetic Control of Programmed Cell Death in the Nematode C. elegans Hilary M. Ellis and H. Robert Horvitz Massachusetts Institute of Technology, Cambridge, MA Cell, 1986, Volume 44, pages 817–829 The Context By the 1960s cell death was recognized as a normal event during animal development, implying that it was a carefully regulated process with specific cells destined to die. The simple nematode C. elegans, which has been a critically important model system in developmental biology, proved to be key to understanding both the regulation and the mechanism of such programmed cell deaths. Microscopic analysis in the 1970s established a complete map of C. elegans development so that the embryonic origin and fate of each cell was known. Importantly, C. elegans development included a very specific pattern of programmed cell deaths. In particular, John Sulston and H. Robert Horvitz reported in 1977 that the development of adult worms (consisting of 959 somatic cells) involved the programmed death of 131 cells out of 1090 that were initially produced. The same cells died in all embryos, indicating that the death of these cells was a normal event during development, with cell death being a specific developmental fate. It was also notable that all of these dying cells underwent a similar series of morphological changes, suggesting that these programmed cell deaths occurred by a common mechanism. Based on these considerations, Horvitz undertook a genetic analysis with the goal of characterizing the mechanism and regulation of programmed cell death during C. elegans development. In the experiments reported in this 1986 paper, Hilary Ellis and Horvitz identified two genes that were required for all of the programmed cell deaths that took place during development of the nematode. The identification and characterization of these genes was a critical first step leading to our current understanding of the molecular biology of apoptosis. The Experiments Cells undergoing programmed cell death in C. elegans can readily be identified as highly refractile cells by microscopic examination, so Ellis and Horvitz were able to use this as an assay to screen for mutant animals in which the normal cell deaths did not occur. To isolate mutants that displayed abnormalities in cell death, they treated nematodes with the chemical mutagen ethyl methanesulfonate, which reacts with DNA. The progeny of approximately 4000 worms were examined to identify dying cells, and two mutant strains were found in which the expected cell deaths did not take place (see figure). Both of these mutant strains harbored recessive mutations of the same gene, which was called ced-3. Further studies indicated that the mutations in ced-3 blocked all of the 131 programmed cell deaths that would normally occur during development. Continuing studies identified an additional mutation that was similar to ced-3 in preventing programmed cell H. Robert Horvitz death. However, this mutation was in a different gene, which was located on a different chromosome than ced-3. This second gene was called ced-4. Similar to mutations in ced-3, recessive mutations in ced-4 were found to block all programmed cell deaths in the worm. The Impact The isolation of C. elegans mutants by Ellis and Horvitz provided the first identification of genes that were involved in the process of programmed cell death. The proteins encoded by the ced-3 and ced-4 genes, as well as by (A) (B) Photomicrographs of a normal worm (A) and a ced-3 mutant (B). Dying cells are highly refractile and are indicated by arrows in panel A. These cells are not present in the mutant animal. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL KEY the ced-9 gene (which was subsequently identified by Horvitz and colleagues), proved to be prototypes of the central regulators and effectors of apoptosis that are highly conserved in evolution. The cloning and sequencing of ced-3 revealed that it was related to a protease that had been previously identified in mammalian cells, and which became the first member of the caspase family. The C. elegans ced-9 5 EXPERIMENT gene was related to the bcl-2 oncogene, first isolated from a human B cell lymphoma, which had the unusual property of inhibiting apoptosis rather than stimulating cell proliferation. And ced-4 was found to encode an adaptor protein related to mammalian Apaf-1, which is required for caspase activation. The identification of these genes in C. elegans thus led the way to understanding the molecular basis of apoptosis, with broad implications both for development and for the maintenance of normal adult tissues. Since abnormalities of apoptosis contribute to a wide variety of diseases, including cancer, autoimmune disease, and neurodegenerative disorders, the seminal findings of Ellis and Horvitz have impacted a wide range of areas in biology and medicine. tion, the normal programmed cell deaths did not take place. A third gene, ced-9, functioned as a negative regulator of apoptosis. If ced-9 was inactivated by mutation, the cells that would normally survive failed to do so. Instead, they also underwent apoptosis, leading to death of the developing animal. Conversely, if ced-9 was expressed at an abnormally high level, the normal programmed cell deaths failed to occur. Further studies indicated that the proteins encoded by these genes acted in a pathway with Ced-4 acting to stimulate Ced-3, and Ced-9 inhibiting Ced-4 (Figure 17.3). Genes related to ced-3, ced-4, and ced-9 have also been identified in Drosophila and mammals and found to encode proteins that represent conserved effectors and regulators of apoptosis induced by a variety of stimuli. Caspases: The Executioners of Apoptosis The molecular cloning and nucleotide sequencing of the ced-3 gene indicated that it encoded a protease, providing the first insight into the molecular mechanism of apoptosis. We now know that Ced-3 is the prototype of a family of more than a dozen proteases, known as caspases because they have cysteine (C) residues at their active sites and cleave after aspartic acid (Asp) residues in their substrate proteins. The caspases are the ultimate effectors or executioners of programmed cell death, bringing about the events of apoptosis by cleaving more than 100 different cell target proteins (Figure 17.4). One key target of the caspases is an inhibitor of a DNase, which when activated is responsible for fragmentation of nuclear DNA. In addition, caspases cleave nuclear lamins, leading to fragmentation of the nucleus; cytoskeletal proteins, leading to disruption of the cytoskeleton, membrane blebbing, and cell fragmentation; and Golgi matrix proteins, leading to fragmentation of the Golgi apparatus. The translocation of phosphatidylserine to the cell surface is also dependent on caspases, although the caspase target(s) responsible for this plasma membrane alteration have not yet been identified. Apoptosis Ced-9 Ced-4 Ced-3 This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. FIGURE 17.3 Programmed cell death in C. elegans Genetic analysis identified three genes that play key roles in programmed cell death during development of C. elegans. Two genes, ced-3 and ced-4, are required for cell death, whereas ced-9 inhibits cell death. The Ced-9 protein acts upstream of Ced-4, which activates Ced-3. UNCORRECTED PAGE PROOFS 6 CHAPTER 17 FIGURE 17.4 Caspase targets Caspases cleave over 100 cellular proteins to induce the morphological alterations characteristic of apoptosis. Caspase targets include an inhibitor of DNase (ICAD), nuclear lamins, cytoskeletal proteins, and Golgi matrix proteins. Caspases ICAD Inhibitor of DNase Nuclear lamins DNA fragmentation Fragmentation of nucleus Cytoskeletal proteins Actin, myosin, a-actinin, tubulin, vimentin Cell fragmentation, membrane blebbing Golgi matrix proteins Fragmentation of Golgi Ced-3 is the only caspase in C. elegans. However, Drosophila and mammals contain families of at least seven caspases, classified as either initiator or effector caspases, that function in a cascade to bring about the events of apoptosis. All caspases are synthesized as inactive precursors that can be converted to the active form by proteolytic cleavage, catalyzed by other caspases. Initiator caspases are activated directly in response to the various signals that induce apoptosis, as discussed later in this chapter. The initiator caspases then cleave and activate the effector caspases, which are responsible for digesting the cellular target proteins that mediate the events of apoptosis (see Figure 17.4). The activation of an initiator caspase therefore starts off a chain reaction of caspase activation leading to death of the cell. Genetic analysis in C. elegans initially suggested that Ced-4 functioned as an activator of the caspase Ced-3. Subsequent studies have shown that Ced4 and its mammalian homolog (Apaf-1) bind to caspases and promote their activation. In mammalian cells, the key initiator caspase (caspase-9) is activated by binding to Apaf-1 in a multisubunit complex called the apoptosome (Figure 17.5). Formation of this complex in mammals also requires Cytochrome c Caspase-9 (initiator caspase) Pro-caspase-3 Apaf-1 Active caspase-3 Effector caspase Apoptosis FIGURE 17.5 Caspase activation The mammalian initiator caspase-9 is activated as a complex with Apaf-1 and cytochrome c in the apoptosome. Caspase-9 then cleaves and activates effector caspases, such as caspase-3. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL cytochrome c, which is released from mitochondria by stimuli that trigger apoptosis (discussed in the following section). Once activated in the apoptosome, caspase-9 cleaves and activates downstream effector caspases, such as caspase-3 and caspase-7, eventually resulting in cell death. Central Regulators of Apoptosis: The Bcl-2 Family The third gene identified as a key regulator of programmed cell death in C. elegans, ced-9, was found to be closely related to a mammalian gene called bcl-2, which was first identified in 1985 as an oncogene that contributed to the development of human B cell lymphomas (cancers of B lymphocytes). In contrast to other oncogene proteins, such as Ras, that stimulate cell proliferation (see Molecular Medicine, Chapter 15), Bcl-2 was found to inhibit apoptosis. Ced-9 and Bcl-2 were thus similar in function, and the role of Bcl2 as a regulator of apoptosis first focused attention on the importance of cell survival in cancer development. As discussed further in the next chapter, we now recognize that cancer cells are generally defective in the normal process of programmed cell death and that their inability to undergo apoptosis is as important as their uncontrolled proliferation in the development of malignant tumors. Mammals encode a family of approximately 20 proteins related to Bcl-2, which are divided into three functional groups (Figure 17.6). Some members of the Bcl-2 family (antiapoptotic family members)—like Bcl-2 itself— function as inhibitors of apoptosis and programmed cell death. Other members of the Bcl-2 family, however, are proapoptotic proteins that act to induce caspase activation and promote cell death. There are two groups of these proapoptotic proteins, which differ in function as well as in their extent of homology to Bcl-2. Bcl-2 and the other antiapoptotic family members share four conserved regions called Bcl-2 homology (BH) domains. One group of proapoptotic family members called the “multidomain” proapoptotic proteins have 3 BH domains (BH1, BH2, and BH3), whereas the second group, the “BH3-only” proteins, have only the BH3 domain. The fate of the cell—life or death—is determined by the balance of activity of proapoptotic and antiapoptotic Bcl-2 family members, which act to regulate one another (Figure 17.7). The multidomain proapoptotic family members, such as Bax and Bak, are the downstream effectors that directly induce apoptosis. They are inhibited by interactions with the antiapoptotic family members, such as Bcl-2. The BH3-only family members are upstream members of the cascade, regulated by the signals that induce cell death (e.g., DNA damage) or cell survival (e.g., growth factors). When activated, the BH3-only family members antagonize the antiapoptotic Bcl-2 family members, activating the multidomain proapoptotic proteins and tipping the balance in favor of caspase activation and cell death. Antiapoptotic Bcl-2 Bcl-xL Proapoptotic multidomain Bax Bak Proapoptotic BH3-only Bid Bad Noxa Puma Bim BH4 BH3 BH1 BH2 BH3 BH1 BH2 FIGURE 17.6 BH3 The Bcl-2 family The Bcl-2 family of proteins is divided into three functional groups. Antiapoptotic proteins (e.g., Bcl-2 and Bcl-xL) have four Bcl-2 homology domains (BH1–BH4). The multidomain proapoptotic proteins (e.g., Bax and Bak) have three homology domains (BH1–BH3), whereas the BH3-only proapoptotic proteins (e.g., Bid, Bad, Noxa, Puma, and Bim) have only one homology domain (BH3). This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. 7 UNCORRECTED PAGE PROOFS 8 CHAPTER 17 FIGURE 17.7 Regulatory interactions between Bcl-2 family members In normal cells, the BH3-only proapoptotic proteins are inactive, and the multidomain proapoptotic proteins are inhibited by interaction with antiapoptotic proteins. Cell death signals activate the BH3-only proteins, which then interact with the antiapoptotic proteins, leading to activation of the multidomain proapoptotic proteins and cell death. Apoptotic cell Normal cell BH3-only protein (inactive) Cell death signal Activation of BH3-only proapoptotic protein BH3-only protein (active) Antiapoptotic protein Multidomain proapoptotic protein Activation of multidomain proapoptotic protein Antiapoptotic protein Apoptosis IAPs were first discovered in virus-infected insect cells as viral proteins that inhibited apoptosis of the host cell. In mammalian cells, members of the Bcl-2 family act at mitochondria, which play a central role in controlling programmed cell death ( Figure 17.8). When activated, Bax and Bak form oligomers in the mitochondrial outer membrane. Formation of these Bax or Bak oligomers leads to the release of cytochrome c f rom the mitochondrial intermembrane space, either by forming pores or by interacting with other mitochondrial outer membrane proteins. The release of cytochrome c from mitochondria then triggers caspase activation. In particular, the key initiator caspase in mammalian cells (caspase-9) is activated by forming a complex with Apaf-1 in the apoptosome. In mammals, formation of this complex also requires cytochrome c. Under normal conditions of cell survival, cytochrome c is localized to the mitochondrial intermembrane space (see Figure 11.10) while Apaf-1 and caspase-9 are found in the cytosol, so caspase-9 remains inactive. Activation of Bax or Bak results in the release of cytochrome c to the cytosol, where it binds to Apaf-1 and triggers apoptosome formation and caspase-9 activation. Caspases are also regulated by a family of proteins called the IAP, for inhibitor of apoptosis, family. Members of the IAP family directly interact with caspases and suppress apoptosis by either inhibiting caspase activity or by targeting caspases for ubiquitination and degradation in the proteasome. IAPs are present in both Drosophila and mammals (but not C. elegans), and regulation of their activity or expression provides another mechanism for controlling apoptosis. Regulation of IAPs is particularly important in Drosophila, where initiator caspases are chronically activated but held in check by IAPs ( Figure 17.9 ). Many signals that induce apoptosis in Drosophila function by activating proteins that inhibit the IAPs, thus leading to caspase activation. In mammalian cells, the permeabilization of mitoThis material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL FIGURE 17.8 The mitochondrial pathway of apoptosis In mammalian cells, many cell death signals induce apoptosis as a result of damage to mitochondria (the intrinsic pathway of apoptosis). When active, the proapoptotic multidomain Bcl-2 family proteins (Bak and Bax) form oligomers in the outer membrane of mitochondria, resulting in the release of cytochrome c from the intermembrane space. Release of cytochrome c leads to the formation of apoptosomes containing Apaf-1 and caspase-9 in which caspase-9 is activated. Caspase-9 then activates downstream caspases, such as caspase-3, by proteolytic cleavage. 9 Death signal Bax chondria by Bax or Bak results not only in the release of cytochrome c but also of IAP inhibitors that may help to stimulate caspase activity. Signaling Pathways that Regulate Apoptosis Programmed cell death is regulated by the integrated activity of a variety of signaling pathways, some acting to induce cell death and others acting to promote cell survival. These signals control the fate of individual cells, so that cell survival or elimination is determined by the needs of the organism as a whole. The pathways that induce apoptosis in mammalian cells are grouped as intrinisic or extrinsic pathways, which differ in their involvement of Bcl-2 family proteins and in the identity of the caspase that initates cell death. One important role of apoptosis is the elimination of damaged cells, so apoptosis is stimulated by many forms of cell stress, including DNA damage, viral infection, and growth factor deprivation. These stimuli activate the intrinsic pathway of apoptosis, which leads to release of cytochrome c from mitochondria and activation of caspase-9 (see Figure 17.8). As illustrated in the following examples, the multiple signals that activate this pathway converge on regulation of the BH3-only members of the Bcl-2 family. DNA damage is a particularly dangerous form of cell stress, because cells with damaged genomes may have suffered mutations that can lead to the development of cancer. DNA damage is thus one of the principal triggers of programmed cell death, leading to the elimination of cells carrying potentially harmful mutations. As discussed in Chapter 16, several cell cycle checkpoints halt cell cycle progression in response to damaged DNA, allowing time for the damage to be repaired. In mammalian cells, a major pathway leading to cell cycle arrest in response to DNA damage is mediated by the transcription factor p53. The ATM and Chk2 protein kinases, which are activated by DNA damage, phosphorylate and stabilize p53. The resulting increase in p53 leads to transcriptional activation of p53 target genes. These include the Cdk inhibitor p21, which inhibits Cdk2/cyclin E complexes, halting cell cycle progression in G1 (see Figure 16.20). However, activation of p53 by DNA damage can also lead to apoptosis (Figure 17.10). The induction of apoptosis by p53 results, at least in part, from transcriptional activation of genes encoding the BH3-only proapoptotic Bcl-2 family members PUMA and Noxa. Increased expression of these BH3-only proteins leads to activation of Bax and Bak, release of cytochrome c from mitochondria, and activation of caspase-9. Thus p53 mediates both cell cycle arrest and apoptosis in response to DNA damage. Whether DNA damage in a given cell leads to apoptosis or reversible cell cycle arrest may depend on the extent of damage and the resulting level of p53 induction, as well as the influence of other life/death signals being received by the cell. Growth factor deprivation is another form of cell stress that activates the intrinsic pathway of apoptosis. In this case, apoptosis is controlled by signaling pathways that promote cell survival by inhibiting apoptosis in This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. Bax Cytochrome c Apaf-1 Apoptosome Caspase-9 Pro-caspase-3 Active caspase-3 Apoptosis UNCORRECTED PAGE PROOFS 10 CHAPTER 17 Figure 17.9 Regulation of caspases by IAPs in Drosophila Both initiator and effector caspases are inhibited by IAPs. Many signals that induce apoptosis in Drosophila function by activating members of a family of proteins (Reaper, Hid, and Grim) that inhibit the IAPs, resulting in caspase activation. Death signal Reaper, Hid, Grim IAPs Initiator and effector caspases Apoptosis DNA damage ATM Chk2 P P Increased levels of p53 p53 PP PP p53p53 response to growth factor stimulation. These signaling pathways control the fate of a wide variety of cells whose survival is dependent on extracellular growth factors or cell-cell interactions. As already noted, a well-characterized example of programmed cell death in development is provided by the vertebrate nervous system. About 50% of neurons die by apoptosis, with the survivors having received sufficient amounts of survival signals from their target cells. These survival signals are polypeptide growth factors related to nerve growth factor (NGF), which induces both neuronal survival and differentiation by activating a receptor protein-tyrosine kinase. Other types of cells are similarly dependent upon growth factors or cell contacts that activate nonreceptor protein-tyrosine kinases associated with integrins. Indeed, most cells in higher animals are programmed to undergo apoptosis unless cell death is actively suppressed by survival signals from other cells. One of the major intracellular signaling pathways responsible for promoting cell survival is initiated by the enzyme PI 3-kinase, which is activated by either protein-tyrosine kinases or G protein-coupled receptors. PI 3-kinase phosphorylates the membrane phospholipid PIP2 to form PIP3, which activates the protein-serine/threonine kinase Akt (see Figures 15.30 and 15.31). Akt then phosphorylates a number of proteins that regulate apoptosis (Figure 17.11). One key substrate for Akt is the proapoptotic BH3only Bcl-2 family member called Bad. Phosphorylation of Bad by Akt creates a binding site for 14-3-3 chaperone proteins that sequester Bad in an inactive form, so phosphorylation of Bad by Akt inhibits apoptosis and promotes cell survival. Bad is similarly phosphorylated by protein kinases of other growth factor-induced signaling pathways, including the Ras/Raf/MEK/ERK pathway, so it serves as a convergent regulator of growth factor signaling in mediating cell survival. Other targets of Akt, including the FOXO transcription factors, also play key roles in cell survival. Phosphorylation of FOXO by Akt creates a binding site for 14-3-3 proteins, which sequester FOXO in an inactive form in the cytoplasm (see Figure 15.32). In the absence of growth factor signaling and Akt activity, FOXO is released from 14-3-3 and translocates to the nucleus, stimulating transcription of proapoptotic genes, including the gene encoding the BH3-only protein, Bim. Akt and its downstream target GSK-3 also regulate other transcription factors with roles in cell survival, including p53 and NF-kB, which control the expression of additional Bcl-2 family members. In addition, the level of the antiapoptotic Bcl-2 family member Mcl-1 may be modulated via translational regulation by both GSK-3 and the mTOR pathway (see Figure 15.33). These multiple effects on members of the Bcl-2 family converge to regulate the intrinsic pathway of apoptosis, PUMA Noxa Apoptosis FIGURE 17.10 Role of p53 in DNA damage-induced apoptosis DNA damage leads to activation of the ATM and Chk2 protein kinases, which phosphorylate and stabilize p53 resulting in rapid increases in p53 levels. The protein p53 then activates transcription of genes encoding the proapoptotic BH3-only proteins PUMA and Noxa, leading to cell death. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL 11 Survival factor Receptor tyrosine kinase Plasma membrane PIP3 PIP2 Akt PDK1 P P P mTOR P mTORC2 PI 3-kinase mTORC1 Active Akt P P GSK-3 Bad FOXO p53 NF-kB Translational regulation Bim FIGURE 17.11 The PI 3-kinase pathway and cell survival Many growth factors that signal cell survival activate receptor protein-tyrosine kinases, leading to activation of PI 3-kinase, formation of PIP3, and activation of the protein kinase, Akt. Akt then phosphorylates a number of proteins that contribute to cell survival. Phosphorylation of the BH3-only protein Bad maintains it in an inactive state, as does phosphorylation of the FOXO transcription factors. In the absence of Akt signaling, activation of Bad promotes apoptosis and activation of FOXO stimulates transcription of another BH3-only protein, Bim. Additional targets of Akt that have been implicated in regulation of apoptosis include the protein kinase GSK-3 and additional transcription factors, such as p53 and NF-kB, both of which are regulated by Akt and GSK-3 phosphorylation. Translational regulation by GSK-3 and by the mTOR pathway (see Figure 15.33) may also affect cell survival. c ontrolling the activation of caspase-9 and cell survival in response to growth factor stimulation. In contrast to the cell stress and growth factor signaling pathways that regulate the intrinsic pathway of apoptosis, some secreted polypeptides activate receptors that induce cell death via the extrinsic pathway of apoptosis. These receptors directly activate a distinct initiator caspase, caspase-8 ( Figure 17.12). The polypeptides that signal cell death by this pathway belong to the tumor necrosis factor (TNF) family. They bind to members of the TNF receptor family, which can induce apoptosis in a variety of cell types. One of the best characterized members of this family is the cell surface receptor called Fas, which plays important roles in controlling cell death in the immune system. For example, apoptosis induced by activation of Fas is responsible for killing target cells of the immune system, such as cancer cells or virus-infected cells, as well as for eliminating excess lymphocytes at the end of an immune response. TNF and related family members consist of three identical polypeptide chains, and their binding induces receptor trimerization. The cytoplasmic This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. Therapies based on a member of the TNF family are under clinical trial for the treatment of certain cancers. UNCORRECTED PAGE PROOFS 12 CHAPTER 17 FIGURE 17.12 Cell death receptors TNF and other cell death receptor ligands consist of three polypeptide chains, so their binding to cell death receptors induces receptor trimerization. Caspase-8 is recruited to the receptor and activated via interaction with adaptor molecules (the extrinsic pathway of apoptosis). Once activated, caspase-8 can directly cleave and activate effector caspases. In addition, caspase-8 cleaves the BH3-only protein Bid, which activates the intrinsic pathway of apoptosis, leading to caspase-9 activation. TNF TNF receptor Cytosol Adaptor Caspase-8 Bid activation Bid Effector caspase activation Bak/Bax Mitochondrion Caspase-9 activation Effector caspase activation portions of the receptors bind adaptor molecules that in turn bind caspase8. This leads to activation of caspase-8, which can then cleave and activate downstream effector caspases. In some cells, caspase-8 activation and subsequent activation of caspases-3 and -7 is sufficient to induce apoptosis directly. In other cells, however, amplification of the signal is needed. This This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL results from caspase-8 cleavage of the proapoptotic BH3-only protein Bid, leading to Bid activation, permeabilization of mitochondria, and activation of caspase-9, thus amplifying the caspase cascade initiated by direct activation of caspase-8 at cell death receptors. Alternative Pathways of Programmed Cell Death Although apoptosis is the most common form of regulated or programmed cell death, recent research has shown that programmed cell death can also occur by alternative, non-apoptotic mechanisms. One of these alternative pathways of regulated cell death is autophagy. As discussed in Chapter 8, autophagy provides a mechanism for the gradual turnover of the cell’s components by the uptake of proteins or organelles into vesicles (autophagosomes) that fuse with lysosomes (see Figure 8.45). In addition, autophagy promotes cell survival under conditions of nutrient deprivation. Under conditions of starvation, activation of autophagy serves to increase the degradation of cellular proteins and organelles, generating energy and allowing their components to be reutilized for essential functions. In other circumstances, however, autophagy provides an alternative to apoptosis as a pathway of cell death. Autophagic cell death does not require caspases and, rather than possessing the distinct morphological features of apoptosis, the dying cells are characterized by an accumulation of lysosomes. Autophagy has been shown to be an important pathway of programmed cell death during salivary gland development in Drosophila and to be induced by infection with some viruses. In addition, autophagy appears to provide an alternative pathway to cell death when apoptosis is blocked. For example, cells of mutant mice lacking Bak and Bax fail to undergo apoptosis in response to stimuli such as DNA damage, as expected since Bak and Bax are required for permeabilization of mitochondria (see Figure 17.8). However, the Bak/Bax-deficient cells die by autophagy instead, suggesting that autophagy may also be activated by cellular stress and provide an alternative to apoptosis under these conditions. It also appears that some forms of necrosis can be a programmed cellular response, rather than simply representing uncontrolled cell lysis as the result of an acute injury. In contrast to unregulated necrosis, these forms of regulated necrotic cell death are induced as a programmed response to stimuli such as infection or DNA damage, which also induce apoptosis. Under these conditions, regulated necrosis may provide an alternative pathway of cell death if apoptosis does not occur. For example, if apoptosis is inhibited, stimulation of the TNF receptor leads to cell death by necrosis. The importance of both autophagy and necrosis as alternatives to apoptosis remains to be fully explored, not only in normal cells but also in diseases such as cancer, heart disease, and neurodegeneration, which involve abnormalities of cell survival. Stem Cells and the Maintenance of Adult Tissues Early development is characterized by the rapid proliferation of embryonic cells, which then differentiate to form the specialized cells of adult tissues and organs. As cells differentiate, their rate of proliferation usually decreases, and most cells in adult animals are arrested in the G0 stage of the cell cycle. However, cells are lost either due to injury or programmed cell death throughout life. In order to maintain a constant number of cells in adult tissues and organs, cell death must be balanced by cell proliferation. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. 13 UNCORRECTED PAGE PROOFS 14 CHAPTER 17 FIGURE 17.13 Skin fibroblasts Scanning electron micrograph of a fibroblast surrounded by collagen fibrils. (© CMEABG-UCBL/Photo Researchers, Inc.) In order to maintain this balance, most tissues contain cells that are able to proliferate as required to replace cells that have died. Moreover, in some tissues a subpopulation of cells divide continuously throughout life to replace cells that have a high rate of turnover in adult animals. Cell death and cell renewal are thus carefully balanced to maintain properly sized and functioning adult tissues and organs. Proliferation of Differentiated Cells Most types of differentiated cells in adult animals are no longer capable of proliferation. If these cells are lost, they are replaced by the proliferation of less differentiated cells derived from self-renewing stem cells, as discussed in the following section. Other types of differentiated cells, however, retain the ability to proliferate as needed to repair damaged tissue throughout the life of the organism. These cells enter the G 0 stage of the cell cycle but resume proliferation as needed to replace cells that have been injured or died. Cells of this type include fibroblasts, which are dispersed in connective tissues where they secrete collagen (Figure 17.13). Skin fibroblasts are normally arrested in G0 but rapidly proliferate if needed to repair damage resulting from a cut or wound. Blood clotting at the site of a wound leads to the release of platelet-derived growth factor (PDGF) from blood platelets. As discussed in Chapter 15, PDGF activates a receptor protein-tyrosine kinase, stimulating both the proliferation of fibroblasts and their migration into the wound where their proliferation and secretion of collagen contributes to repair and regrowth of the damaged tissue. The endothelial cells that line blood vessels (Figure 17.14) are another type of fully differentiated cell that remains capable of proliferation. Proliferation of endothelial cells allows them to form new blood vessels as needed for repair and regrowth of damaged tissue. Endothelial cell proliferation and the resulting formation of blood capillaries is triggered by a growth factor (vascular endothelial growth factor or VEGF) produced by This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL 15 FIGURE 17.14 Endothelial cells Electron micrograph of a capillary. The capillary is lined by a single endothelial cell surrounded by a thin basal lamina. (© Dr. Don W. Fawcett/Visuals Unlimited.) Capillary lumen Endothelial cell cells of the tissue that the new capillaries will invade. The production of VEGF is in turn triggered by a lack of oxygen, so the result is a regulatory system in which tissues that have a low oxygen supply resulting from insufficient circulation stimulate endothelial cell proliferation and recruit new capillaries (Figure 17.15). Smooth muscle cells, which form the walls of larger blood vessels (e.g., arteries) as well as the contractile portions of the digestive and respiratory tracts and other internal organs, are also capable of resuming proliferation in response to growth factor stimulation. In contrast, differentiated skeletal and cardiac muscle cells are no longer able to divide. Tissue deprived of oxygen VEGF Endothelial cells proliferate in response to VEGF Capillary FIGURE 17.15 Proliferation of endothelial cells Endothelial cells are stimulated to proliferate by vascular endothelial growth factor (VEGF). VEGF is secreted by cells deprived of oxygen, leading to the outgrowth of new capillaries into tissues lacking an adequate blood supply. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS 16 CHAPTER 17 FIGURE 17.16 Liver regeneration Liver cells are normally arrested in G0 but resume proliferation to replace damaged tissue. If two-thirds of the liver of a rat is surgically removed, the remaining cells proliferate to regenerate the entire liver in a few days. Liver Removal of two-thirds of liver Proliferation of remaining cells Regeneration of liver The epithelial cells of some internal organs are also able to proliferate to replace damaged tissue. A striking example is provided by liver cells, which are normally arrested in the G0 phase of the cell cycle. However, if large numbers of liver cells are lost (e.g., by surgical removal of part of the liver), the remaining cells are stimulated to proliferate to replace the missing tissue ( Figure 17.16 ). For example, surgical removal of two-thirds of the liver of a rat is followed by rapid proliferation of the remaining cells, leading to regeneration of the entire liver within a few days. Stem Cells Most fully differentiated cells in adult animals, however, are no longer capable of cell division. Nonetheless, they can be replaced by the proliferation of a subpopulation of less differentiated self-renewing cells called stem cells that are present in most adult tissues. Because they retain the capacity to proliferate and replace differentiated cells throughout the lifetime of an animal, stem cells play a critical role in the maintenance of most tissues and organs. The key property of stem cells is that they divide to produce one daughter cell that remains a stem cell and one that divides and differentiates (Figure 17.17). Because the division of stem cells produces new stem cells as well as differentiated daughter cells, stem cells are selfrenewing populations that can serve as a source for the production of differentiated cells throughout life. The role of stem cells is particularly evident in the case of several types of differentiated cells, including blood cells, sperm, epithelial cells of the skin, and epithelial cells lining the digestive tract—all of which have short life spans and must be replaced by continual cell proliferation in adult animals. In all of these cases, the fully differentiated cells do not themselves proliferate; instead, they are continually renewed by the proliferation of stem cells that then differentiate to maintain a stable number of differentiated cells. Stem cells have also been identified in a variety of other adult tissues, including skeletal muscle and the nervous system, where they function to replace damaged tissue. Stem cells were first identified in the hematopoietic (blood-forming) system by Ernest McCulloch and James Till in 1961 in experiments showing that single cells derived from mouse bone marrow could proliferate and give rise to multiple differentiated types of blood cells. Hematopoietic stem cells are well-characterized and the production of blood cells provides a good example of the role of stem cells in maintaining differentiated cell populations. There are several distinct types of blood cells with specialized functions: erythrocytes (red blood cells) that transport O2 and CO2; granulocytes and macrophages, which are phagocytic cells; platelets (which are fragments of megakaryocytes) that function in blood coagulation; and lymphocytes that are responsible for the immune response. All these cells have limited life spans ranging from less than a day to a few months, and all are derived from the same population of hematopoietic stem cells. More than This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL Stem cell Self renewal Proliferation Differentiation Differentiated cells FIGURE 17.17 Stem cell proliferation Stem cells divide to form one daughter cell that remains a stem cell and a second that proliferates and then differentiates. 100 billion blood cells are lost every day in humans, and must be continually produced from hematopoietic stem cells in the bone marrow (Figure 17.18). Descendants of the hematopoietic stem cell continue to proliferate and undergo several rounds of division as they become committed to specific differentiation pathways that are determined by growth factors that channel precursor cells along specific pathways of blood cell differentiation. Once they become fully differentiated, blood cells cease proliferation, so the maintenance of differentiated blood cell populations is dependent on continual division of the self-renewing hematopoietic stem cell. The intestine provides an excellent example of stem cells in the selfrenewal of an epithelial tissue. The intestine is lined by a single layer of epithelial cells that are responsible for the digestion of food and absorption of nutrients. These intestinal epithelial cells are exposed to an extraordinarily harsh environment and have a lifetime of only a few days before they die by apoptosis and are shed into the digestive tract. Renewal of the intestinal epithelium is therefore a continual process throughout life. New cells This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. 17 UNCORRECTED PAGE PROOFS 18 CHAPTER 17 Hematopoietic stem cell Myeloid Reticulocyte Megakaryocyte Lymphoid Monocyte Erythrocyte Platelets Neutrophil Macrophage Eosinophil Basophil B lymphocyte T lymphocyte Granulocytes FIGURE 17.18 Formation of blood cells All of the different types of blood cells develop from a hematopoietic stem cell in the bone marrow. The precursors of differentiated cells undergo several rounds of cell division before they differentiate. are derived from the continuous but slow division of stem cells at the bottom of intestinal crypts (Figure 17.19). The stem cells give rise to a population of transit-amplifying cells, which divide rapidly and occupy about two-thirds of the crypt. The transit-amplifying cells proliferate for three to four cell divisions and then differentiate into the three cell types of the colon surface epithelium: absorptive epithelial cells and two types of secretory cells, called goblet cells and enteroendocrine cells. The small intestine also contains a fourth cell type, Paneth cells, which secrete antibacterial agents. Each crypt contains approximately six self-renewing stem cells, which can give rise to all of the different types of cells in the intestinal epithelium. Stem cells are also responsible for continuous renewal of the skin and hair. Like the lining of the intestine, the skin and hair are exposed to a harsh external environment—including ultraviolet radiation from sunlight—and are continuously renewed throughout life. The skin consists of three major cell lineages: the epidermis, hair follicles, and sebaceous glands, which This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL (A) Surface epithelium Absorptive epithelial cells Cell shedding 19 Goblet Enterocell endocrine cell Crypt Transitamplifying cells Transitamplifying cells Stem cell (B) Surface epithelium Stem cell Crypt FIGURE 17.19 Renewal of the intestinal epithelium (A) Colon epithelial cells are renewed by division of stem cells located at the bottom of the intestinal crypt. The stem cell gives rise to a population of transit-amplifying cells, which occupy about two-thirds of the crypt and undergo three to four divisions before differentiating into the three cell types of the surface epithelium (absorptive epithelial cells, goblet cells, and enteroendocrine cells). The surface epithelial cells continually undergo apoptosis and are shed into the intestinal lumen. (B) Micrograph of a colon crypt and surface epithelium. Proliferating cells are stained with antibody against a cell cycle protein (brown nuclei). (From F. Radtke and H. Clevers, 2005. Science 307: 1904.) release oils that lubricate the skin surface. Each of these three cell populations is maintained by their own stem cells (Figure 17.20). The epidermis is a multilayered epithelium, which is undergoing continual cell renewal. In humans, the epidermis turns over every two weeks, with cells being sloughed from the surface. These cells are replaced by epidermal stem cells, which reside in a single basal layer. The epidermal stem cells give rise to transit-amplifying cells, which undergo three to six divisions before differentiating and moving outward to the surface of the skin. The stem cells responsible for producing hair reside in a region of the hair follicle called the bulge. The bulge stem cells give rise to transit-amplifying matrix cells, This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS 20 CHAPTER 17 Epidermis Hair follicle Hair shaft Cell loss Sebaceous gland stem cells Sebaceous gland Differentiation Transitamplifying cells Bulge (hair follicle stem cells) Epidermal stem cell FIGURE 17.20 Stem cells of the skin The epidermis consists of multiple layers of epithelial cells. Cells from the surface are continually lost and replaced by epidermal stem cells in the basal layer. The stem cells give rise to transit-amplifying cells, which undergo several divisions in the basal layer before differentiating and moving to the surface of the skin. Stem cells of hair follicles reside in a region beneath the sebaceous gland called the bulge, and distinct stem cells of the sebaceous gland reside at its base. which proliferate and differentiate to form the hair shaft. Finally, a distinct population of stem cells resides at the base of the sebaceous gland. It is notable that, if the skin is injured, stem cells of the bulge can also give rise to epidermis and sebaceous glands, demonstrating their activity as multipotent stem cells from which both skin and hair can be derived. Skeletal muscle provides an example of the role of stem cells in the repair of damaged tissue, in contrast to the continual cell renewal just described in the hematopoietic system, intestinal epithelium, and skin. Skeletal muscle is composed of large multinucleated cells (muscle fibers) formed by cell fusion during development (see Figure 12.21). Although skeletal muscle is normally a stable tissue with little cell turnover, it is able to regenerate rapidly in response to injury or exercise. This regeneration is mediated by proliferation of satellite cells, which are the stem cells of adult muscle. Satellite cells are located beneath the basal lamina of muscle fibers (Figure 17.21). They are normally quiescent, arrested in the G0 phase of the cell cycle, but are activated to proliferate in response to injury or exercise. Once activated, the satellite cells give rise to progeny that undergo several divisions and then differentiate and fuse to form new muscle fibers. The continuing capacity of skeletal muscle to regenerate throughout life is due to selfrenewal of the satellite stem cell population. Stem cells have also been found in many other adult tissues, including the brain and heart, and it is possible that most—if not all—tissues contain stem cells with the potential of replacing cells that are lost during the lifetime of the organism. It appears that stem cells reside within distinct microenvironments, called niches, which provide the environmental signals that maintain stem cells throughout life and control the balance between their self-renewal and differentiation. Stem cells are rare in adult mammalian tissues, however, so the precise identification of stem cells and their niches represents a major challenge in the field of stem cell biology. For example, although the role of stem cells in maintenance of the intestinal epithelium has long been recognized, the intestinal stem cells at the base of the crypt (see Figure 17.19) were only recently identified by studies of Hans This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL (A) (B) 21 Nucleus of muscle fiber Satellite cell One muscle fiber (muscle cell) Myofibril Nucleus of muscle fiber FIGURE 17.21 Muscle satellite cells (A) The stem cells of skeletal muscle are the satellite cells, located beneath the basal lamina of muscle fibers. (B) Electron micrograph showing a satellite cell and the nucleus of a muscle fiber. (From S. Chargé and M. Rudnicki, 2003. Physiol. Rev. 84: 209; courtesy of Sophie Chargé and Michael Rudnicki.) Satellite cell Plasma membrane Basal lamina Myofibril Clever and his colleagues in 2007. Signaling by the Wnt pathway (see Figure 15.44) plays a major role in controlling the proliferation of these stem cells, and it is thought that Wnt polypeptides secreted by fibroblasts of the underlying connective tissue are responsible for intestinal stem cell maintenance. Wnt signaling is also involved in regulation of several other types of stem cells, including stem cells of the skin and hematopoietic system. In addition, signaling by the TGF-b, Hedgehog, and Notch pathways (see Figures 15.41, 15.43, and 15.45) play important roles in stem cell regulation, although the precise roles of these factors in regulating different types of stem cells within their distinct niches remains to be understood. Medical Applications of Adult Stem Cells The ability of adult stem cells to repair damaged tissue clearly suggests their potential utility in clinical medicine. If these stem cells could be isolated and propagated in culture, they could in principal be used to replace damaged tissue and treat a variety of disorders, such as diabetes or degenerative diseases like muscular dystrophy, Parkinson’s or Alzheimer’s disease. In some cases, the use of stem cells derived from adult tissues may be the optimal approach for such stem cell therapies, although the use of embryonic stem cells (discussed in the next section of this chapter) is likely to provide a more versatile approach to treatment of a wider variety of disorders. A well-established clinical application of adult stem cells is hematopoietic stem cell transplantation (or bone marrow transplantation), which plays an important role in the treatment of a variety of cancers. As discussed in Chapter 18, most cancers are treated by chemotherapy with drugs that kill rapidly dividing cells by damaging DNA or inhibiting DNA replication. These drugs do not act selectively against cancer cells but are also This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS 22 CHAPTER 17 FIGURE 17.22 Hematopoietic stem cell transplantation A cancer patient is treated with high doses of chemotherapy, which effectively kill tumor cells but normally would not be tolerated because of potentially lethal damage to the hematopoietic system. This damage is then repaired by transplantation of new hematopoietic stem cells. Stored umbilical cord blood from an unrelated donor can also be used as a source of hematopoietic stem cells for transplantation. High dose chemotherapy Hematopoietic stem cell transplant Tumor Cancer patient Drugs kill tumor cells Toxicity to hematopoietic system Restoration of hematopoietic system toxic to those normal tissues that are dependent on continual renewal by stem cells, such as blood, skin, hair, and the intestinal epithelium. The hematopoietic stem cells are among the most rapidly dividing cells of the body, so the toxic effects of anticancer drugs on these cells frequently limit the effectiveness of chemotherapy in cancer treatment. Hematopoietic stem cell transplantation provides an approach to bypassing this toxicity, thereby allowing the use of higher drug doses to treat the patient’s cancer more effectively. In this procedure, the patient is treated with high doses of chemotherapy that would normally not be tolerated because of toxic effects on the hematopoietic system (Figure 17.22). The potentially lethal damage is repaired, however, by transferring new hematopoietic stem cells (obtained either from bone marrow or peripheral blood) to the patient following completion of chemotherapy, so that a normal hematopoietic system is restored. In some cases, the stem cells are obtained from the patient prior to chemotherapy, stored, and then returned to the patient once chemotherapy is completed. However, it is important to ensure that these cells are not contaminated with cancer cells. Alternatively, the stem cells to be transplanted can be obtained from a healthy donor (usually a close relative) whose tissue type closely matches the patient. In addition to their use in cancer treatment, hematopoietic stem cell transfers are used to treat patients with diseases of the hematopoietic system, such as aplastic anemia, hemoglobin disorders, and immune deficiencies. Epithelial stem cells have also found clinical application in the form of skin grafts that are used to treat patients with burns, wounds, and ulcers. One approach to these procedures is to culture epidermal skin cells to form an epithelial sheet, which can then be transferred to the patient. Because the patient’s own skin can be used for this procedure, it eliminates the potential complication of graft rejection by the immune system. The possibilities of using adult stem cells for similar replacement therapies of other diseases, including diabetes, Parkinson’s disease, and muscular dystrophies, are being actively pursued. However, these clinical applications of adult stem cells are limited by the difficulties in isolating and culturing the appropriate stem cell populations. Embryonic Stem Cells and Therapeutic Cloning While adult stem cells are difficult to isolate and culture, it is relatively straightforward to isolate and propagate the stem cells of early embryos (embryonic stem cells). These cells can be grown indefinitely as pure stem cell populations while maintaining the ability to give rise to all of the differentiated cell types of adult organisms. Consequently, there has been an enormous interest in embryonic stem cells from the standpoints of both basic science and clinical applications. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS C ELL DEATH AND CELL RENEWAL KEY 23 EXPERIMENT Culture of Embryonic Stem Cells Isolation of a Pluripotent Cell Line from Early Mouse Embryos Cultured in Medium Conditioned by Teratocarcinoma Stem Cells Gail R. Martin University of California at San Francisco Proceedings of the National Academy of Science, USA, 1981, Volume 78, pages 7634–7638 The Context The cells of early embryos are unique in their ability to proliferate and differentiate into all of the types of cells that make up the tissues and organs of adult animals. In 1970 it was found that early mouse embryos frequently developed into tumors if they were removed from the uterus and transplanted to an abnormal site. These tumors, called teratocarcinomas, contained cells that were capable of forming an array of different tissues as they grew within the animal. In addition, cells from teratocarcinomas (called embryonal carcinoma cells) could be isolated and grown in tissue culture. These cells resembled normal embryo cells and could be induced to differentiate into a variety of cell types in culture. Some embryonal carcinoma cells could also participate in normal development of a mouse if they were injected into early mouse embryos (blastocysts) that were then implanted into a foster mother. The ability of embryonal carcinoma cells to differentiate into a variety of (A) (B) cell types and to participate in normal mouse development suggested that these tumor-derived cells might be closely related to normal embryonic stem cells. However, the events that occurred during the establishment of teratocarcinomas in mice were unknown. Gail Martin hypothesized that the embryonal carcinoma cells found in teratocarcinomas were essentially normal embryo cells that proliferated abnormally simply because, when they were removed from the uterus and transplanted to an abnormal site, they did not receive the appropriate signals to induce normal differentiation. Based on this hypothesis, she attempted to culture cells from mouse embryos with the goal of isolating normal embryonic stem cell lines. Her experiments, together with similar work by Martin Evans and Matthew Kaufman (Establishment in culture of pluripotential cells from mouse embryos, Nature, 1981, 292: 154–156), demonstrated that stem cells could be cultured directly from (C) Gail R. Martin normal mouse embryos. The isolation of these embryonic stem cell lines paved the way to genetic manipulation and analysis of mouse development, as well as to the possible use of human embryonic stem cells in transplantation therapy. The Experiments Based on the premise that embryonal carcinoma cells were derived from normal embryonic stem cells, Martin attempted to culture cells from normal mouse blastocysts. Starting with cells from approximately 30 embryos, she initially isolated four colonies of growing cells after a week of culture. These cells could be repeatedly passaged into mass cultures, and new cell lines could be reproducibly derived when the Embryonic stem cells differentiate in culture to a variety of cell types, including neuronlike cells (A), endodermal cells (B), and cartilage (C). (Continued on next page.) This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS 24 CHAPTER 17 KEY EXPERIMENT Culture of Embryonic Stem Cells (continued ) experiment was repeated with additional mouse embryos. The cell lines derived from normal embryos (embryonic stem cells) closely resembled the embryonal carcinoma cells derived from tumors. Most importantly, the embryonic stem cells could be induced to differentiate in culture into a variety of cell types, including endodermal cells, cartilage, and neuron-like cells (see figure). Moreover, if the embryonic stem cells were injected into a mouse, they formed tumors containing multiple differentiated cell types. It thus appeared that embryonic stem cell lines, which retained the ability to differentiate into a wide array of cell types, could be established in culture from normal mouse embryos. The Impact The establishment of embryonic stem cell lines has had a major impact on studies of mouse genetics and development as well as opening new possibilities for the treatment of a variety of human diseases. Subsequent experiments demonstrated that embryonic stem cells could participate in normal mouse development following their injection into mouse embryos. Since gene transfer techniques could be used to introduce or mutate genes in cultured embryonic stem cells, these cells have been used to investigate the role of a variety of genes in mouse development. As discussed in Chapter 4, any gene of interest can be inactivated in embryonic stem cells by homologous recombination with a cloned DNA, and the role of that gene in mouse development can then be determined by introducing the altered embryonic stem cells into mouse embryos. In 1998 two groups of researchers developed the first lines of human embryonic stem cells. Because of the proliferative and differentiative capacity of these cells, they offer the hope of providing new therapies for the treatment of a variety of diseases. Although a number of technical problems and ethical concerns need to be addressed, transplantation therapies based on the use of embryonic stem cells may provide the best hope for eventual treatment of diseases such as Parkinson’s, Alzheimer’s, diabetes, and spinal cord injuries. Embryonic Stem Cells Embryonic stem cells were first cultured from mouse embryos in 1981 (Figure 17.23). They can be propagated indefinitely in culture and, if reintroduced into early embryos, are able to give rise to cells in all tissues of the mouse. Thus they retain the capacity to develop into all of the different types of cells in adult tissues and organs (referred to as pluripotency). In addition, they can be induced to differentiate into a variety of different types of cells in culture. As discussed in Chapter 4, mouse embryonic stem cells have been an important experimental tool in cell biology because they can be used to introduce altered genes into mice (see Figure 4.36). Moreover, they provide an outstanding model system for studying the molecular and cellular events associated with embryonic cell differentiation, so embryonic stem cells have long been of considerable interest to cell and developmental biologists. Interest in these cells reached a new peak of intensity, however, in 1998 when two groups of researchers reported the isolation of stem cells (A) Embryo FIGURE 17.23 Culture of mammalian embryonic stem cells (A) Embryonic stem cells are cultured from the inner cell mass of an early embryo (blastocyst). (B) Scanning electron micrograph of cultured embryonic stem cells. (Yorgos Nikas/Photo Researchers, Inc.) Culture of embryonic stem cells (B) Inner cell mass This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL Undifferentiated ES cells maintained in LIF Removal of LIF FIGURE 17.24 Differentiation of embryonic stem cells Mouse embryonic stem (ES) cells are maintained in the undifferentiated state in the presence of LIF. If LIF is removed from the culture medium, the cells aggregate to form embryoid bodies and then differentiate into a variety of cell types. Embryoid bodies Cell differentiation Blood cells Epithelial cells Adipocytes Smooth muscle cells Nerve cells from human embryos, raising the possibility of using embryonic stem cells in clinical transplantation therapies. Mouse embryonic stem cells are grown in the presence of a growth factor called LIF (for leukemia inhibitory factor), which signals through the JAK/STAT pathway (see Figure 15.40) and is required to maintain these cells in their undifferentiated state (Figure 17.24). If LIF is removed from the medium, the cells aggregate into structures that resemble embryos (embryoid bodies) and then differentiate into a wide range of cell types, including neurons, adipocytes, blood cells, epithelial cells, vascular smooth muscle cells, and even beating heart muscle cells. Human embryonic stem cells do not require LIF but are similarly maintained in the undifferentiated state by other growth factors, which are not yet fully characterized. Importantly, embryonic stem cells can be directed to differentiate along specific pathways by the addition of appropriate growth factors to the culture medium. It may thus be possible to derive populations of specific types of cells, such as heart cells or nerve cells, for transplantation therapy. For example, methods have been developed to direct the differentiation of both mouse and human embryonic stem cells into cardiomyocytes, which have been used to repair heart damage resulting from myocardial infarction in mice. Likewise, considerable progress has been made in directing the differentiation of embryonic stem cells to neurons, which have been used for transplantation therapy in rodent models of Parkinson’s disease and spinal cord injury, and to insulin-producing pancreatic cells, which have been used for therapy in mouse models of diabetes. A great deal of current research is therefore focused on the development of culture conditions to promote the differentiation of embryonic stem cells along specific pathways, thereby producing populations of differentiated cells that can be used for transplantation therapy of a variety of diseases. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. 25 Heart muscle cells UNCORRECTED PAGE PROOFS 26 CHAPTER 17 (A) (B) Unfertilized egg Adult somatic cell Remove egg chromosomes Transfer adult nucleus to enucleated egg Culture to early embryo Blastocyst Implant in foster mother FIGURE 17.25 Cloning by somatic cell nuclear transfer (A) The nucleus of an adult somatic cell is transferred to an unfertilized egg from which the normal egg chromosomes have been removed (an enucleated egg). The egg is then cultured to an early embryo and transferred to a foster mother, who then gives birth to a clone of the donor of the adult nucleus. (B) Dolly (the adult sheep, left) was the first cloned mammal. She is shown with her lamb, Bonnie, who was produced by normal reproduction. (Photograph by Roddy Field; courtesy of T. Wakayama and R. Yanagimachi.) Somatic Cell Nuclear Transfer Clone T he isolation of human embryonic stem cells in 1998 followed the first demonstration that the nucleus of an adult mammalian cell could give rise to a viable cloned animal. In 1997 Ian Wilmut and his colleagues initiated a new era of regenerative medicine with the cloning of Dolly the sheep (Figure 17.25). Dolly arose from the nucleus of a mammary epithelial cell that was transplanted into an unfertilized egg in place of the normal egg nucleus—a process called somatic cell nuclear transfer. It is interesting to note that this type of experiment was first carried out in frogs in the 1950s. The fact that it took over 40 years before it was successfully performed in mammals attests to the technical difficulty of the procedure. Since the initial success of Wilmut and his colleagues, transfer of nuclei from adult somatic cells into enucleated eggs has been used to create cloned offspring of a variety of mammalian species, including sheep, mice, pigs, cattle, goats, rabbits, and cats. However, cloning by somatic cell nuclear transfer in mammals remains an extremely inefficient procedure, such that only 1–3% of embryos generally give rise to live offspring. Animal cloning by somatic cell nuclear transfer, together with the properties of embryonic stem cells, opens the possibility of therapeutic cloning (Figure 17.26). In therapeutic cloning, a nucleus from an adult human cell would be transferred to an enucleated egg, which would then be used to produce an early embryo in culture. Embryonic stem cells could then be cultured from the cloned embryo and used to generate appropriate types of differentiated cells for transplantation therapy. The major advantage provided by therapeutic cloning is that the embryonic stem cells derived by this procedure would be genetically identical to the recipient of the transplant, who was the donor of the adult somatic cell nucleus. This bypasses the barrier of the immune system in rejecting the transplanted tissue. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS CELL DEATH AND CELL RENEWAL FIGURE 17.26 Therapeutic cloning In therapeutic cloning, the nucleus of a patient’s cell would be transferred to an enucleated egg, which would be cultured to an early embryo. Embryonic stem cells would then be derived, differentiated into the desired cell type and transplanted back into the patient. The transplanted cells would be genetically identical to the recipient (who was the donor of the adult nucleus), so complications of immune rejection would be avoided. Adult somatic cell from patient Unfertilized egg Remove egg chromosomes Transfer nucleus to enucleated egg The possibility of therapeutic cloning provides the most general approach to treatment of the wide variety of devastating disorders for which stem cell transplantation therapy could be applied. However, although some success has been achieved in animal models, there remain major obstacles that would need to be overcome before therapeutic cloning could be applied to humans. Substantial improvements would be needed to overcome the low efficiency with which embryos are generated by somatic cell nuclear transfer. In addition, therapeutic cloning by somatic cell nuclear transfer raises ethical concerns, not only with respect to the possibility of cloning human beings (reproductive cloning ), but also with respect to the destruction of embryos that serve as the source of embryonic stem cells. These concerns may be alleviated by recent advances in reprogramming somatic cells to a pluripotent state resembling embryonic stem cells. Culture to early embryo Blastocyst Induced Pluripotent Stem Cells Given both the technical and ethical difficulties in the derivation of embryonic stem cells by somatic cell nuclear transfer, a major advance in the field has come from studies demonstrating that adult somatic cells can be directly converted to pluripotent stem cells in culture. This circumvents the need for derivation of embryos and provides a direct mechanism for converting somatic cells to stem cells which, like embryonic stem cells, have the potential of developing into all tissues of an organism. The conversion (or reprogramming) of somatic cells to pluripotent stem cells was first reported by Kazutoshi Takahashi and Shinya Yamanaka in 2006. They found that mouse fibroblasts could be reprogrammed to cells resembling embryonic stem cells (called induced pluripotent stem cells) by the action of only four transcription factors introduced by retroviral gene transfer ( Figure 17.27 ). Subsequent studies have shown that induced pluripotent stem cells, like embryonic stem cells, are capable of differentiating into all cell types when introduced into early mouse embryos. That the action of only four key transcription factors is sufficient to reprogram adult somatic cells to pluripotent stem cells is a remarkable finding, which raises a number of intriguing questions as to the transcriptional programs that control cell fate. Importantly, further research has also demonstrated that adult human fibroblasts can be reprogrammed to pluripotency by a similar procedure. Thus, it is now possible to convert skin cells from a patient directly to induced pluripotent stem cells in culture, providing a new route to the derivation of pluripotent stem This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. 27 Culture embryonic stem cells Differentiate to desired cell type (e.g., neurons) Transplant back to patient UNCORRECTED PAGE PROOFS 28 CHAPTER 17 Adult mouse fibroblast Infection with retroviruses carrying genes for Oct3/4, Sox2, Klf4 and c-Myc Induced pluripotent stem cell FIGURE 17.27 Induced pluripotent stem cells Adult mouse fibroblasts in culture are converted to pluripotent stem cells by infection with retroviral vectors (see Figure 4.34) carrying genes for four transcription factors: Oct3/4, Sox2, Klf4, and c-Myc. cells for use in transplantation therapy. However, problems still remain to be addressed. Some of the transcription factors (e.g., c-Myc) used to reprogram fibroblasts to pluripotent stem cells can act as oncogenes to cause cancer, although it may be possible to substitute these potentially harmful transcription factors with others that do not have oncogenic potential. Additionally, the retroviral vectors that have been used to introduce genes into fibroblasts can themselves cause harmful mutations leading to cancer development, so alternative methods of gene transfer will need to be developed for safe therapeutic applications. Nonetheless, overcoming these difficulties may enable induced pluripotent stem cells to be readily established and used for patient-specific transplantation therapy. SUMMARY KEY TERMS COMPANION WEBSITE Visit the website that accompanies The Cell (www.sinauer.com/cooper5e) for animations, videos, quizzes, problems, and other review material. programmed cell death, necrosis, apoptosis PROGRAMMED CELL DEATH The Events of Apoptosis: Programmed cell death plays a key role in both the maintenance of adult tissues and embryonic development. In contrast to the accidental death of cells from an acute injury, programmed cell death takes place by the active process of apoptosis. Apoptotic cells and cell fragments are then efficiently removed by phagocytosis. Genes responsible for the regulation and execution of apoptosis were initially identified by genetic analysis of C. elegans. See Website Animation 17.1 caspase, apoptosome Caspases: The Executioners of Apoptosis: The caspases are a family of proteases that are the effectors of apoptosis. Caspases are classified as either initiator or effector caspases, and both function in a cascade leading to cell death. In mammalian cells, the major initiator caspase is activated in a complex called the apoptosome, which also requires cytochrome c released from mitochondria. Bcl-2, IAP Central Regulators of Apoptosis: the Bcl-2 Family: Members of the Bcl2 family are central regulators of caspase activation and apoptosis. Some members of the Bcl-2 family function to inhibit apoptosis (antiapoptotic) whereas others act to promote apoptosis (proapoptotic). Signals that control programmed cell death alter the balance between proapoptotic and antiapoptotic Bcl-2 family members, which regulate one another. In mammalian cells, proapoptotic Bcl-2 family members act at mitochondria, where they promote the release of cytochrome c, leading to caspase activation. Caspases are also regulated directly by inhibitory IAP proteins. This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS C ELL DEATH AND CELL RENEWAL SUMMARY 29 KEY TERMS Signaling Pathways that Regulate Apoptosis: A variety of signaling pathways regulate apoptosis by controlling the expression or activity of proapoptotic members of the Bcl-2 family. These pathways include DNA damage-induced activation of the tumor suppressor p53, growth factorstimulated activation of PI 3-kinase/Akt signaling, and activation of death receptors by polypeptides that induce programmed cell death. p53, PI 3-kinase, Akt, tumor necrosis factor (TNF) Alternative Pathways of Programmed Cell Death: Autophagy and regulated necrosis provide alternatives to apoptosis for induction of programmed cell death. autophagy STEM CELLS AND THE MAINTENANCE OF ADULT TISSUES Proliferation of Differentiated Cells: Most cells in adult animals are arrested in the G0 stage of the cell cycle. A few types of differentiated cells, including skin fibroblasts, endothelial cells, smooth muscle cells, and liver cells are able to resume proliferation as required to replace cells that have been lost because of injury or cell death. Stem Cells: Most differentiated cells do not themselves proliferate but can be replaced via the proliferation of stem cells. Stem cells divide to produce one daughter cell that remains a stem cell and another that divides and differentiates. Stem cells have been identified in a wide variety of adult tissues, including the hematopoietic system, skin, intestine, skeletal muscle, brain, and heart. stem cell, niche Medical Applications of Adult Stem Cells: The ability of stem cells to repair damaged tissue suggests their potential use in clinical medicine. Adult stem cells are used to repair damage to the hematopoietic system in hematopoietic stem cell transplantation, and epidermal stem cells can be used for skin grafts. However, clinical applications of adult stem cells are limited by difficulties in isolating and culturing these cells. hematopoietic stem cell transplantation, bone marrow transplantation EMBRYONIC STEM CELLS AND THERAPEUTIC CLONING Embryonic Stem Cells: Embryonic stem cells are cultured from early embryos. They can be readily grown in the undifferentiated state in culture while retaining the ability to differentiate into a wide variety of cell types, so they may offer considerable advantages over adult stem cells for many clinical applications. embryonic stem cell, pluripotency Somatic Cell Nuclear Transfer: Mammals have been cloned by somatic cell nuclear transfer in which the nucleus of an adult somatic cell is transplanted into an enucleated egg. This opens the possibility of therapeutic cloning in which embryonic stem cells would be derived from a cloned embryo and used for transplantation therapy of the donor of the adult nucleus. Although many obstacles need to be overcome, the possibility of therapeutic cloning holds great promise for the development of new treatments for a variety of devastating diseases. somatic cell nuclear transfer, therapeutic cloning, reproductive cloning Induced Pluripotent Stem Cells: Adult somatic cells can be converted to pluripotent stem cells in culture by four key transcription factors, potentially providing an alternative to embryonic stem cells for transplantation therapy. induced pluripotent stem cell This material cannot be copied, reproduced, manufactured, or disseminated in any form without express written permission from the publisher. © 2009 Sinauer Associates, Inc. UNCORRECTED PAGE PROOFS 30 CHAPTER 17 Questions 1. Why is cell death via apoptosis more advantageous to multicellular organisms than cell death via acute injury? been mutated such that Akt no longer phosphorylates it. How would expression of this mutant affect cell survival? 2. What molecular mechanisms regulate caspase activity? 7. How would expression of siRNA targeted against 14-3-3 proteins affect apoptosis? forms large channels in the mitochondrial outer membrane, releasing proteins from the intermembrane space into the cytoplasm. How will treatment with this polypeptide affect mammalian cells in culture? 8. You are considering treatment of a leukemic patient with TNF. Upon further analysis you determine that the leukemic cells have an inactivating mutation of caspase-8. Will treatment with TNF be an effective therapy for this patient? 11. Many adult tissues contain terminally differentiated cells that are incapable of proliferation. However, these tissues can regenerate following damage. What gives these tissues their regenerative capabilities? 9. How would siRNA against Ced-3 affect the development of C. elegans? 12. What is the critical property of stem cells? 10. 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