- Email: [email protected]
What Are Stem Cells?
C H A P T E R
3 What Are Stem Cells?
O U T L I N E What are the Key Properties of Stem Cells?
46
Totipotency, Pluripotency, and Embryonic Stem Cells
47
Multipotency, Unipotency and Adult Stem Cells
50
Cell Division and Aging: the Role of Telomerase
51
The Relation Between Cell Division and Differentiation: Epigenetics 52 Epigenetics in Stem Cells
Stem Cells Scientific Facts and Fiction
55
45
Copyright Ó 2011 Elsevier Inc. All rights reserved.
46
3. WHAT ARE STEM CELLS?
The common definition of a stem cell is a cell that can divide to give rise to a new copy of itself and at least one other specialized (differentiated) cell type. Although this broad definition provides a useful framework, much still remains to be discovered about the different types of stem cells in the body, their common as well as unique properties, and how and for what applications they can be used in research, medical practice, biotechnology, and pharmacology.
WHAT ARE THE KEY PROPERTIES OF STEM CELLS? In addition to being able to divide and produce new copies of themselves (self-renew), stem cells can also differentiate, or specialize, into other cell types. If the stem cell is able to form all cell types of the embryo and adult, including germ cells (eggs and sperm) and the extra-embryonic structures such as placenta, it is considered totipotent. A fertilized egg cell, for example, is totipotent, as are some of the early blastomeres resulting from cleavage (or division) of the fertilized egg; these cells are not stem cells however, since they do not self-renew. If a stem cell can do all of these things but is unable to form the extra-embryonic structures (such as parts of the placenta) then it is called pluripotent. Embryonic stem cells are pluripotent. All other stem cells found in specialized tissues of the fetus or adult are multipotent, meaning that they are able to form many but not all tissue cells of the body, or are unipotent, able to form just one other cell type. Spermatogonia for instance are unipotent and are only able to form sperm. In order to be able to divide without losing the stem cell pool for later use, a stem cell is capable of multiplying by dividing in two, but after each cell division at least one of the two daughter cells retains the original stem cell properties. The daughter cell that loses stem cell properties then becomes fully differentiated: a specialized cell that produces all of the proteins required for its proper function within the tissue or organ it belongs to. It can at best divide only a few times but more often not at all; this is for example the case for (at least most) brain cells. To simplify discussion, it is easiest conceptually to divide stem cells into two types: embryonic stem cells and adult stem cells. Embryonic stem cells are derived from a very early embryo, and adult stem cells are found in postnatal tissues, not only of the body but also the umbilical cord. Although much controversy still exists about the potency of adult stem cells and their ability to form different cell types, adult stem cells are generally considered to be either multipotent or unipotent. In the adult body in vivo there are no totipotent or pluripotent stem cells. This does not mean that the artificial conditions of culture cannot convert (“reprogram”) some adult stem cells, like spermatogonia in the testis, into stem cells with pluripotent properties. We will return to this issue of
TOTIPOTENCY, PLURIPOTENCY, AND EMBRYONIC STEM CELLS
47
reprogramming in culture later. Under normal circumstances, however, a differentiated cell cannot change back into a stem cell since differentiation is in principle a one way process. Fully differentiated cells can at best only divide a limited number of times to give rise to daughter cells with the same characteristics, while differentiation into other cell types is not an option. Similarly, a multipotent or unipotent adult stem cell in an organ or tissue, like the pancreas for example, cannot normally revert to the pluripotent state of an embryonic cell. By contrast, embryonic stem cells are by definition pluripotent, and can in principle develop into all cell types in the human body. During embryonic development, as all the different new cell types emerge and tissues are formed, most cells gradually lose their stem cell characteristics and with this their ability to differentiate into many different directions. This differentiation process starts when the totipotent fertilized egg moves on into the pluripotent state of the inner cell mass of a blastocyst stage embryo, then subsequently into specialized cells and into the multi- or unipotent stem cells of specific organs or tissues. These later stem cells are also known as progenitor cells. Progenitor cells can divide and are predestined to differentiate only to the cell types that are needed for the proper function of their own specific organ or tissue. The biology underlying the unidirectionality of differentiation is based on epigenetic modifications and mechanisms discussed earlier (see Chapter 1). In normal development, an ever-increasing number of genes are more or less permanently altered by epigenetic modifications which guarantee the stability of the differentiated state and limit the ability of cells to divide indefinitely. These changes were thought to be irreversible, although the birth of cloned animals (Chapter 6), the derivation of induced pluripotent stem (iPS) cells from adult cells (Chapter 5), and identification of cancer stem cells (Chapter 9) have demonstrated this is not always the case. In general, however, the epigenetic modifications that take place in normal development reduce the differentiation options for the individual cells and their ability to proliferate.
TOTIPOTENCY, PLURIPOTENCY, AND EMBRYONIC STEM CELLS Our body is made up of some 220 different types of cells, all of which are descendants of a single fertilized egg. The DNA or genome of the egg contains the entire program for the development of the embryo. The fertilized egg develops into a blastocyst stage embryo within a few days of fertilization. At this point, the first division of function between groups of cells takes place; the earliest totipotent cells have disappeared and are replaced by an inner population of
48
3. WHAT ARE STEM CELLS?
pluripotent cells and an outer layer of multipotent cells. This all takes place after three cell divisions when the embryo consists of only eight identical cells. At this stage, one single cell can actually be removed and used, for example for prenatal genetic testing to diagnose specific disease-causing mutations in the embryo. The embryo itself, now consisting only of the remaining seven cells, can develop normally and be transferred to the uterus where, if all goes well, a normal baby will be born. The missing cell is replaced without a problem and prenatal genetic testing thus provides a means of selecting genetically normal from abnormal embryos in families where specific, often untreatable, genetic diseases are prevalent. The procedure demonstrates that the cells of the embryo at this point are still entirely totipotent with unlimited plasticity. At the next embryonic stage, the blastocyst is formed and the distinction between pluripotency and multipotency becomes clearer. The innermost population of cells make up the inner cell mass, from which the entire embryo is formed. The outermost cells form the trophectoderm, which gives rise to parts of the placenta and umbilical cord. Cells from the inner cell mass have become pluripotent and can thus differentiate into all the different cell types that make up the body but can no longer contribute for example to placenta tissue. In these cells, almost all genes are still ready to be used for differentiation in a specific direction. This pluripotent period lasts for only a short time, as cells quickly start to differentiate as development proceeds. When embryonic cells are removed from the inner cell mass (say from an albino mouse that has a white coat color) and injected into a blastocyst
A human egg (left panel) that has been fertilized in the laboratory develops to a multicellular embryo (middle panel). One cell is removed from the embryo using a glass pipette (right panel) and used to investigate whether a disease-causing mutation is present in the genomic DNA. In the right panel the embryo is held by a so-called holding pipette (on the left of the embryo) while a cell is carefully aspirated (or removed) with another pipette on the right. When the gene analysis is complete and the embryo found to be normal, it can be placed in the woman’s womb and develop into a healthy baby.
TOTIPOTENCY, PLURIPOTENCY, AND EMBRYONIC STEM CELLS
49
from another mouse (say with a black coat color), the resulting pups that are born will have coats with black and white patches, with cellular descendants of the injected inner cell mass cells present in all tissues and organs. These pups are then said to be chimeric. Such experiments have provided the evidence that cells of the inner cell mass indeed have the capacity to form all cell types in an adult mouse. If the cells in the inner cell mass are carefully removed from an embryo and cultured in a Petri dish in a laboratory, an embryonic stem cell line can be created. Under the right culture conditions these cells will continue to divide outside the embryo for an unlimited period of time (in contrast to their temporary presence in the embryo) and retain their pluripotent characteristics. If cells from an embryonic stem cell line derived from an albino mouse are injected into a blastocyst as above, chimeric pups with black and white coats will also be born, just as for freshly isolated inner cell mass cells, even if the embryonic stem cells have been grown in culture for more than 20 years. Indeed, a remarkable demonstration that embryonic stem cells do not lose their pluripotent characteristics, even after very long periods of culture.
A preserved and stuffed chimeric mouse generated at Vanderbilt University in Nashville, Tennessee. A chimeric mouse can develop after introduction of embryonic stem cells into a blastocyst stage embryo. If the embryo is then transferred to the uterus of a recipient female mouse, the embryo can implant in the uterine wall and develop to term. The embryonic stem cells can participate in the formation of the embryo. If the blastocyst used came from albino (white coat color) parents and the stem cells originally came from a black mouse embryo, the resulting chimeric mouse will have a black and white coat and it will be quite clear which parts originate from the embryonic stem cells.
50
3. WHAT ARE STEM CELLS?
MULTIPOTENCY, UNIPOTENCY AND ADULT STEM CELLS A curiosity of early development is that even though most cells differentiate and take up their specialized functions after the pluripotent blastocyst stage, a few cells somehow succeed in maintaining, or regaining, stem cell properties. In an adult, most or possibly all organs and tissues contain a small reserve of what we call adult stem cells. The most well-known of this type of stem cell are the blood stem cells in the bone marrow. These organ stem cells usually sit quietly doing little but have the capacity to divide and differentiate if necessary, for example following damage to other cells in the organ or tissue. These adult stem cells are multipotent and the spectrum of cell types they can form is generally limited to those normally present in the organ from which they derive. Stem cells from the bone marrow can for instance form all cells that constitute the blood, but cannot form nerve cells, intestinal cells, or insulin-producing cells. The ability to differentiate to different cell types is even more limited in stem cells that can only give rise to one type of cell: as already mentioned for example spermatogonial stem cells that can only form sperm cells. Adult stem cells in an organism are clearly different from embryonic stem cells as they maintain their ability to form one stem cell and one differentiated daughter cell at each division over the course of an entire lifetime e 70e80 years in humans. Exceptionally, two differentiated daughter cells may form resulting in depletion of the stem cell pool.
Bone marrow that resides within the large bones can be isolated and cells it contains cultured in vitro. In this picture, pig bone marrow cells that have been cultured can be seen.
CELL DIVISION AND AGING: THE ROLE OF TELOMERASE
51
CELL DIVISION AND AGING: THE ROLE OF TELOMERASE If normal cells are cultured outside the body in a Petri dish, they can usually divide a limited number of times when placed under optimal growth conditions, but thereafter cell division ceases and cells become quiescent. This is called senescence. Stem cells, some cancer cells, and some genetically or epigenetically modified cells are the only types of cells that can multiply indefinitely outside the body. The enzyme telomerase plays a key role in this process. Chromosomes in the nucleus of the cell have so-called telomeres at each end of the DNA chains, made up of well-defined, repeating nucleotide sequences. These telomeres function as protective caps for the chromosomes but after every cell division they are shortened. Once they are “used up”, errors can occur in the chromosomal DNA during the next cell division, most often leading to death of the cell. Thus, the shorter the telomere, the “older” the cell. Before the telomeric sequence becomes so short that errors in the rest of the chromosomal DNA ensue, most normal cells respond by committing “suicide” through a special cellular mechanism known as apoptosis. Unlike normal body cells, embryonic stem cells synthesize a relatively large amount of telomerase. This enzyme restores the original size of the telomeres after each cell division, so the chromosomal caps remain intact, uniquely providing stem cells with eternal life .
A dog testis. Sperm cells in the testis are continuously replenished by spermatogonial stem cells.
52
3. WHAT ARE STEM CELLS?
THE RELATION BETWEEN CELL DIVISION AND DIFFERENTIATION: EPIGENETICS Although the nucleotide sequence of the genome of a fertilized egg is not different from that of differentiated cells, the fertilized egg does differ significantly from differentiated cells in one specific respect: epigenetic regulation (Chapter 1). As mentioned earlier, totipotent and pluripotent cells in an early embryo are free to choose in which direction they will differentiate. These cells have yet to acquire cell-specific functions: for example, they do not produce insulin in response to glucose in the way a pancreas cell would. What they are good at is dividing: about once per day (or even more frequently in mice) each cell divides into two daughter cells such that the
EPIGENETIC MODIFICATION OF THE SECOND X CHROMOSOME IN FEMALE CELLS: IMPRINTING
3.1
Women have two female sex chromosomes (XX), and men have one X and one Y chromosome (XY) in each cell. As with the autosomal chromosomes, females have two copies of every gene present on the X chromosomes, while males have only one X chromosome and therefore only one copy of each gene on this chromosome. Two active copies of a gene could lead to overproduction of the corresponding protein, causing major problems during embryo development and postnatal life. A wellknown example is Down syndrome, caused by an extra, third, copy of chromosome 21; the genes on that chromosome are present in threefold (“triploid”) instead of the usual twofold (diploid). Nature has found a solution to this problem in normal individuals by permanently deactivating one of the two X chromosomes very early in embryo development, in whichever cell two X chromosomes are found. As a result, the DNA remains present in the cell, but cannot be actively used any more e this process is called imprinting. Other proteins ensure that when the cell divides, X chromosome inactivity is reproduced in the two daughter cells. The X chromosome to be inactivated is randomly picked and this takes place during early embryonic development. As a consequence, in approximately half of the cells of a normal early embryo, the paternal X chromosome is switched off, while in the other half the maternal X chromosome is inactivated. This means that throughout the whole body, around half of the cells make use of the father’s X chromosome, while the other half have this X chromosome permanently inactivated.
THE RELATION BETWEEN CELL DIVISION AND DIFFERENTIATION: EPIGENETICS
3.1 EPIGENETIC MODIFICATION OF THE SECOND X CHROMOSOME IN FEMALE C E L L S : I M P R I N T I N G (c on t’ d )
Mutant male
- Mutant paternal allele silenced by imprinting - Normal maternal allele active - Normal phenotype
Normal
- Normal paternal allele silenced by imprinting - Normal maternal allele active - Normal phenotype
Mutant female
- Normal paternal allele silenced by imprinting - Mutant maternal allele active - Mutant phenotype
Imprinting is the mechanism by which expression of certain genes is activated or repressed depending on whether the chromosome originates from the sperm (father) or from the egg (mother). When a gene is imprinted so that it is only expressed from the paternal chromosome, a child from a father with a mutation in this gene that would lead to disease is not affected provided the mother does not carry a mutation. The gene from the father is imprinted and, although mutated, is not expressed. Since the gene from the maternal side is intact, the child will not be affected (left). Similarly, if neither parent carries the mutation, the child will also not be affected (middle). However, when the father is normal but the mother carries the mutation, the child will be affected. Although the father’s gene contains a normal gene, its expression is repressed because of the imprinting. The gene is only expressed from the maternal side that carries the mutation (right).
53
54
3. WHAT ARE STEM CELLS?
embryo grows in size and cell number. As the cells start to differentiate and specialize, the ability of individual cells to proliferate decreases. A general rule is that the more differentiated a cell is, the less capable it is of dividing. This applies not only to cells in the living embryo or adult but also to cells in culture in the laboratory. Each cell type has its own function and to fulfil this function it needs its “own” group of special genes to be active. As cells become more specialized, the number of genes they use becomes limited to this essential set; other genes not required for the specialist function are blocked by
In general there are two different ways by which stem cells can divide. At the top, stem cells (yellow) are shown that divide into two identical new stem cells. This is called symmetric cell division. The stem cells can also divide in such a way that one cell remains a stem cell and the other differentiates to give rise to a specialized cell (blue) that is not a stem cell. This is called asymmetric cell division. At the bottom of the illustration, stem cells dividing into one new stem cell and one specialized cell are shown. In this way, the pool of stem cells remains unchanged.
EPIGENETICS IN STEM CELLS
55
dedicated chemical modification of the DNA and surrounding histone proteins that together make up the chromatin. These are the epigenetic modifications, and on the DNA this is seen as addition of methyl groups to certain C nucleotides in the promoter regions of the genes to be inactivated. In addition, the histone proteins that form molecular complexes with the DNA can be chemically altered, thereby changing the accessibility of the DNA to other proteins. With advancing differentiation, the genes required for the complex process of cell division are similarly blocked for use. Thus, fully differentiated and specialized cells, such as brain and heart muscle cells, are left inherently with a limited ability to divide and can thus dedicate all of their time to their assigned function in the body. Another consequence of these epigenetic changes that come with differentiation is that a cell differentiated in one direction cannot simply change into another differentiated cell type: the repertoire of genes that other cell types have available has been made inaccessible. Less differentiated cells, like multipotent stem cells, present in, for example, heart, pancreas and bone marrow, can use their genome somewhat more extensively and choose to become one of several possible subtypes of cells in the organ they are part of, while in addition they can still multiply to some extent. It will be evident that multipotent stem cells in an organ, such as the blood stem cells and mesenchymal stem cells in the bone marrow, are cells with even fewer epigenetic limitations. Finally, stem cells present in the early embryo are characteized by minimal DNA blocking.
EPIGENETICS IN STEM CELLS It has become clear that epigenetic changes, reflected in specific DNA methylation patterns, are often abnormal in embryonic stem cells that have been in culture in a laboratory for a long time. This implies that different embryonic stem cell lines are in this respect not necessarily identical, which probably explains why different cell lines often exhibit slightly different characteristics, for example in growth rate or the ease with which they differentiate in a certain direction. One reason for this is that during each cell division there is a chance of introducing an abnormality into the DNA being replicated which would therefore also be present in the two daughter cells. This way cells can accidentally acquire additional features, like mutations or abnormally methylated genes, which provide them with a growth advantage. Since rapidly proliferating cells tend to dominate and overgrow more slowly proliferating cells, they are gradually selected out during culture at the expense of cells with the original characteristics. In this way, after multiple passages in culture, when only the very distant relatives of the original cells are present, slightly different cell lines may evolve.
56
3. WHAT ARE STEM CELLS?
3.2
WHAT’S SO STRANGE ABOUT PLANARIA?
The regenerative capability of human tissues and organs is limited. A superficial wound will heal, but limbs or organs that are lost do not regrow. This is not true for all animal species. Less complex organisms often have remarkable regenerative capacities. Research into these species can teach us a great deal about the regenerative capability of tissues and pluripotency of cells. Planaria are kinds of small flatworms that have been investigated very extensively in the past. Planaria is a collective name for a variety of flatworms, including Schmidtea mediterranea and Dugesia japonica. Planaria are simple organisms, but they are composed of cells from three germ cell layers e just like mammals e namely the ectoderm, endoderm and mesoderm. They therefore have a nervous system and eyes (formed from ectoderm), muscles (from mesoderm) and a digestive tract (from endoderm). Planaria flatworms have been studied mostly because of their remarkable regenerative capacity. If a Planaria flatworm is cut into two parts, each half will grow to form a complete flatworm. If the head is separated from the body, or the body is cut into a number of pieces, each piece will form a new worm with a head and tail, each animal being genetically identical to the original animal e they are clones of the original animal. What is most interesting is that a piece of Planaria without a head will form a new head, but not an extra tail; Planaria without a tail will form a tail and no head. The cells that form new structures apparently also have positional information, know where they are in the Planaria’s body and somehow know exactly what to make. Planaria’s regenerative success is caused by a special group of cells with a large nucleus and little cytoplasm, called neoblasts. About 25e30% of Planaria cells are neoblasts. Little is known about these cells; it is still not clear whether they are formed from a self-renewing stem cell population or from specialized cells that de-differentiate. Neoblasts are probably totipotent, although it cannot be excluded that the neoblast population is intrinsically heterogeneous and that different neoblasts exist for various cell types. In intact Planaria, cell division takes place continuously, and new cells e formed from neoblasts e replace old cells that have served their function. If a Planaria is damaged, neoblasts migrate to the site of damage. Once there, they divide to increase the local number of neoblasts. These cells then differentiate into the required cell types and replace the damaged tissue. Exactly which signals make the neoblasts decide to
EPIGENETICS IN STEM CELLS
3.2
WHAT’S SO STRANGE ABOUT P L A N A R I A ? ( co nt ’d)
migrate to the wound, while preventing them from differentiating into different cell types within the organism, and how they obtain the exact positional information, are questions that remain unanswered. A better understanding of these mysteries may contribute to our knowledge of how human stem cells work.
If a Planaria flatworm is cut into several pieces, every part can reform (or regenerate) the missing piece so that within a few days each individual part has grown out to become a new flatworm.
57
3 What Are Stem Cells?
O U T L I N E What are the Key Properties of Stem Cells?
46
Totipotency, Pluripotency, and Embryonic Stem Cells
47
Multipotency, Unipotency and Adult Stem Cells
50
Cell Division and Aging: the Role of Telomerase
51
The Relation Between Cell Division and Differentiation: Epigenetics 52 Epigenetics in Stem Cells
Stem Cells Scientific Facts and Fiction
55
45
Copyright Ó 2011 Elsevier Inc. All rights reserved.
46
3. WHAT ARE STEM CELLS?
The common definition of a stem cell is a cell that can divide to give rise to a new copy of itself and at least one other specialized (differentiated) cell type. Although this broad definition provides a useful framework, much still remains to be discovered about the different types of stem cells in the body, their common as well as unique properties, and how and for what applications they can be used in research, medical practice, biotechnology, and pharmacology.
WHAT ARE THE KEY PROPERTIES OF STEM CELLS? In addition to being able to divide and produce new copies of themselves (self-renew), stem cells can also differentiate, or specialize, into other cell types. If the stem cell is able to form all cell types of the embryo and adult, including germ cells (eggs and sperm) and the extra-embryonic structures such as placenta, it is considered totipotent. A fertilized egg cell, for example, is totipotent, as are some of the early blastomeres resulting from cleavage (or division) of the fertilized egg; these cells are not stem cells however, since they do not self-renew. If a stem cell can do all of these things but is unable to form the extra-embryonic structures (such as parts of the placenta) then it is called pluripotent. Embryonic stem cells are pluripotent. All other stem cells found in specialized tissues of the fetus or adult are multipotent, meaning that they are able to form many but not all tissue cells of the body, or are unipotent, able to form just one other cell type. Spermatogonia for instance are unipotent and are only able to form sperm. In order to be able to divide without losing the stem cell pool for later use, a stem cell is capable of multiplying by dividing in two, but after each cell division at least one of the two daughter cells retains the original stem cell properties. The daughter cell that loses stem cell properties then becomes fully differentiated: a specialized cell that produces all of the proteins required for its proper function within the tissue or organ it belongs to. It can at best divide only a few times but more often not at all; this is for example the case for (at least most) brain cells. To simplify discussion, it is easiest conceptually to divide stem cells into two types: embryonic stem cells and adult stem cells. Embryonic stem cells are derived from a very early embryo, and adult stem cells are found in postnatal tissues, not only of the body but also the umbilical cord. Although much controversy still exists about the potency of adult stem cells and their ability to form different cell types, adult stem cells are generally considered to be either multipotent or unipotent. In the adult body in vivo there are no totipotent or pluripotent stem cells. This does not mean that the artificial conditions of culture cannot convert (“reprogram”) some adult stem cells, like spermatogonia in the testis, into stem cells with pluripotent properties. We will return to this issue of
TOTIPOTENCY, PLURIPOTENCY, AND EMBRYONIC STEM CELLS
47
reprogramming in culture later. Under normal circumstances, however, a differentiated cell cannot change back into a stem cell since differentiation is in principle a one way process. Fully differentiated cells can at best only divide a limited number of times to give rise to daughter cells with the same characteristics, while differentiation into other cell types is not an option. Similarly, a multipotent or unipotent adult stem cell in an organ or tissue, like the pancreas for example, cannot normally revert to the pluripotent state of an embryonic cell. By contrast, embryonic stem cells are by definition pluripotent, and can in principle develop into all cell types in the human body. During embryonic development, as all the different new cell types emerge and tissues are formed, most cells gradually lose their stem cell characteristics and with this their ability to differentiate into many different directions. This differentiation process starts when the totipotent fertilized egg moves on into the pluripotent state of the inner cell mass of a blastocyst stage embryo, then subsequently into specialized cells and into the multi- or unipotent stem cells of specific organs or tissues. These later stem cells are also known as progenitor cells. Progenitor cells can divide and are predestined to differentiate only to the cell types that are needed for the proper function of their own specific organ or tissue. The biology underlying the unidirectionality of differentiation is based on epigenetic modifications and mechanisms discussed earlier (see Chapter 1). In normal development, an ever-increasing number of genes are more or less permanently altered by epigenetic modifications which guarantee the stability of the differentiated state and limit the ability of cells to divide indefinitely. These changes were thought to be irreversible, although the birth of cloned animals (Chapter 6), the derivation of induced pluripotent stem (iPS) cells from adult cells (Chapter 5), and identification of cancer stem cells (Chapter 9) have demonstrated this is not always the case. In general, however, the epigenetic modifications that take place in normal development reduce the differentiation options for the individual cells and their ability to proliferate.
TOTIPOTENCY, PLURIPOTENCY, AND EMBRYONIC STEM CELLS Our body is made up of some 220 different types of cells, all of which are descendants of a single fertilized egg. The DNA or genome of the egg contains the entire program for the development of the embryo. The fertilized egg develops into a blastocyst stage embryo within a few days of fertilization. At this point, the first division of function between groups of cells takes place; the earliest totipotent cells have disappeared and are replaced by an inner population of
48
3. WHAT ARE STEM CELLS?
pluripotent cells and an outer layer of multipotent cells. This all takes place after three cell divisions when the embryo consists of only eight identical cells. At this stage, one single cell can actually be removed and used, for example for prenatal genetic testing to diagnose specific disease-causing mutations in the embryo. The embryo itself, now consisting only of the remaining seven cells, can develop normally and be transferred to the uterus where, if all goes well, a normal baby will be born. The missing cell is replaced without a problem and prenatal genetic testing thus provides a means of selecting genetically normal from abnormal embryos in families where specific, often untreatable, genetic diseases are prevalent. The procedure demonstrates that the cells of the embryo at this point are still entirely totipotent with unlimited plasticity. At the next embryonic stage, the blastocyst is formed and the distinction between pluripotency and multipotency becomes clearer. The innermost population of cells make up the inner cell mass, from which the entire embryo is formed. The outermost cells form the trophectoderm, which gives rise to parts of the placenta and umbilical cord. Cells from the inner cell mass have become pluripotent and can thus differentiate into all the different cell types that make up the body but can no longer contribute for example to placenta tissue. In these cells, almost all genes are still ready to be used for differentiation in a specific direction. This pluripotent period lasts for only a short time, as cells quickly start to differentiate as development proceeds. When embryonic cells are removed from the inner cell mass (say from an albino mouse that has a white coat color) and injected into a blastocyst
A human egg (left panel) that has been fertilized in the laboratory develops to a multicellular embryo (middle panel). One cell is removed from the embryo using a glass pipette (right panel) and used to investigate whether a disease-causing mutation is present in the genomic DNA. In the right panel the embryo is held by a so-called holding pipette (on the left of the embryo) while a cell is carefully aspirated (or removed) with another pipette on the right. When the gene analysis is complete and the embryo found to be normal, it can be placed in the woman’s womb and develop into a healthy baby.
TOTIPOTENCY, PLURIPOTENCY, AND EMBRYONIC STEM CELLS
49
from another mouse (say with a black coat color), the resulting pups that are born will have coats with black and white patches, with cellular descendants of the injected inner cell mass cells present in all tissues and organs. These pups are then said to be chimeric. Such experiments have provided the evidence that cells of the inner cell mass indeed have the capacity to form all cell types in an adult mouse. If the cells in the inner cell mass are carefully removed from an embryo and cultured in a Petri dish in a laboratory, an embryonic stem cell line can be created. Under the right culture conditions these cells will continue to divide outside the embryo for an unlimited period of time (in contrast to their temporary presence in the embryo) and retain their pluripotent characteristics. If cells from an embryonic stem cell line derived from an albino mouse are injected into a blastocyst as above, chimeric pups with black and white coats will also be born, just as for freshly isolated inner cell mass cells, even if the embryonic stem cells have been grown in culture for more than 20 years. Indeed, a remarkable demonstration that embryonic stem cells do not lose their pluripotent characteristics, even after very long periods of culture.
A preserved and stuffed chimeric mouse generated at Vanderbilt University in Nashville, Tennessee. A chimeric mouse can develop after introduction of embryonic stem cells into a blastocyst stage embryo. If the embryo is then transferred to the uterus of a recipient female mouse, the embryo can implant in the uterine wall and develop to term. The embryonic stem cells can participate in the formation of the embryo. If the blastocyst used came from albino (white coat color) parents and the stem cells originally came from a black mouse embryo, the resulting chimeric mouse will have a black and white coat and it will be quite clear which parts originate from the embryonic stem cells.
50
3. WHAT ARE STEM CELLS?
MULTIPOTENCY, UNIPOTENCY AND ADULT STEM CELLS A curiosity of early development is that even though most cells differentiate and take up their specialized functions after the pluripotent blastocyst stage, a few cells somehow succeed in maintaining, or regaining, stem cell properties. In an adult, most or possibly all organs and tissues contain a small reserve of what we call adult stem cells. The most well-known of this type of stem cell are the blood stem cells in the bone marrow. These organ stem cells usually sit quietly doing little but have the capacity to divide and differentiate if necessary, for example following damage to other cells in the organ or tissue. These adult stem cells are multipotent and the spectrum of cell types they can form is generally limited to those normally present in the organ from which they derive. Stem cells from the bone marrow can for instance form all cells that constitute the blood, but cannot form nerve cells, intestinal cells, or insulin-producing cells. The ability to differentiate to different cell types is even more limited in stem cells that can only give rise to one type of cell: as already mentioned for example spermatogonial stem cells that can only form sperm cells. Adult stem cells in an organism are clearly different from embryonic stem cells as they maintain their ability to form one stem cell and one differentiated daughter cell at each division over the course of an entire lifetime e 70e80 years in humans. Exceptionally, two differentiated daughter cells may form resulting in depletion of the stem cell pool.
Bone marrow that resides within the large bones can be isolated and cells it contains cultured in vitro. In this picture, pig bone marrow cells that have been cultured can be seen.
CELL DIVISION AND AGING: THE ROLE OF TELOMERASE
51
CELL DIVISION AND AGING: THE ROLE OF TELOMERASE If normal cells are cultured outside the body in a Petri dish, they can usually divide a limited number of times when placed under optimal growth conditions, but thereafter cell division ceases and cells become quiescent. This is called senescence. Stem cells, some cancer cells, and some genetically or epigenetically modified cells are the only types of cells that can multiply indefinitely outside the body. The enzyme telomerase plays a key role in this process. Chromosomes in the nucleus of the cell have so-called telomeres at each end of the DNA chains, made up of well-defined, repeating nucleotide sequences. These telomeres function as protective caps for the chromosomes but after every cell division they are shortened. Once they are “used up”, errors can occur in the chromosomal DNA during the next cell division, most often leading to death of the cell. Thus, the shorter the telomere, the “older” the cell. Before the telomeric sequence becomes so short that errors in the rest of the chromosomal DNA ensue, most normal cells respond by committing “suicide” through a special cellular mechanism known as apoptosis. Unlike normal body cells, embryonic stem cells synthesize a relatively large amount of telomerase. This enzyme restores the original size of the telomeres after each cell division, so the chromosomal caps remain intact, uniquely providing stem cells with eternal life .
A dog testis. Sperm cells in the testis are continuously replenished by spermatogonial stem cells.
52
3. WHAT ARE STEM CELLS?
THE RELATION BETWEEN CELL DIVISION AND DIFFERENTIATION: EPIGENETICS Although the nucleotide sequence of the genome of a fertilized egg is not different from that of differentiated cells, the fertilized egg does differ significantly from differentiated cells in one specific respect: epigenetic regulation (Chapter 1). As mentioned earlier, totipotent and pluripotent cells in an early embryo are free to choose in which direction they will differentiate. These cells have yet to acquire cell-specific functions: for example, they do not produce insulin in response to glucose in the way a pancreas cell would. What they are good at is dividing: about once per day (or even more frequently in mice) each cell divides into two daughter cells such that the
EPIGENETIC MODIFICATION OF THE SECOND X CHROMOSOME IN FEMALE CELLS: IMPRINTING
3.1
Women have two female sex chromosomes (XX), and men have one X and one Y chromosome (XY) in each cell. As with the autosomal chromosomes, females have two copies of every gene present on the X chromosomes, while males have only one X chromosome and therefore only one copy of each gene on this chromosome. Two active copies of a gene could lead to overproduction of the corresponding protein, causing major problems during embryo development and postnatal life. A wellknown example is Down syndrome, caused by an extra, third, copy of chromosome 21; the genes on that chromosome are present in threefold (“triploid”) instead of the usual twofold (diploid). Nature has found a solution to this problem in normal individuals by permanently deactivating one of the two X chromosomes very early in embryo development, in whichever cell two X chromosomes are found. As a result, the DNA remains present in the cell, but cannot be actively used any more e this process is called imprinting. Other proteins ensure that when the cell divides, X chromosome inactivity is reproduced in the two daughter cells. The X chromosome to be inactivated is randomly picked and this takes place during early embryonic development. As a consequence, in approximately half of the cells of a normal early embryo, the paternal X chromosome is switched off, while in the other half the maternal X chromosome is inactivated. This means that throughout the whole body, around half of the cells make use of the father’s X chromosome, while the other half have this X chromosome permanently inactivated.
THE RELATION BETWEEN CELL DIVISION AND DIFFERENTIATION: EPIGENETICS
3.1 EPIGENETIC MODIFICATION OF THE SECOND X CHROMOSOME IN FEMALE C E L L S : I M P R I N T I N G (c on t’ d )
Mutant male
- Mutant paternal allele silenced by imprinting - Normal maternal allele active - Normal phenotype
Normal
- Normal paternal allele silenced by imprinting - Normal maternal allele active - Normal phenotype
Mutant female
- Normal paternal allele silenced by imprinting - Mutant maternal allele active - Mutant phenotype
Imprinting is the mechanism by which expression of certain genes is activated or repressed depending on whether the chromosome originates from the sperm (father) or from the egg (mother). When a gene is imprinted so that it is only expressed from the paternal chromosome, a child from a father with a mutation in this gene that would lead to disease is not affected provided the mother does not carry a mutation. The gene from the father is imprinted and, although mutated, is not expressed. Since the gene from the maternal side is intact, the child will not be affected (left). Similarly, if neither parent carries the mutation, the child will also not be affected (middle). However, when the father is normal but the mother carries the mutation, the child will be affected. Although the father’s gene contains a normal gene, its expression is repressed because of the imprinting. The gene is only expressed from the maternal side that carries the mutation (right).
53
54
3. WHAT ARE STEM CELLS?
embryo grows in size and cell number. As the cells start to differentiate and specialize, the ability of individual cells to proliferate decreases. A general rule is that the more differentiated a cell is, the less capable it is of dividing. This applies not only to cells in the living embryo or adult but also to cells in culture in the laboratory. Each cell type has its own function and to fulfil this function it needs its “own” group of special genes to be active. As cells become more specialized, the number of genes they use becomes limited to this essential set; other genes not required for the specialist function are blocked by
In general there are two different ways by which stem cells can divide. At the top, stem cells (yellow) are shown that divide into two identical new stem cells. This is called symmetric cell division. The stem cells can also divide in such a way that one cell remains a stem cell and the other differentiates to give rise to a specialized cell (blue) that is not a stem cell. This is called asymmetric cell division. At the bottom of the illustration, stem cells dividing into one new stem cell and one specialized cell are shown. In this way, the pool of stem cells remains unchanged.
EPIGENETICS IN STEM CELLS
55
dedicated chemical modification of the DNA and surrounding histone proteins that together make up the chromatin. These are the epigenetic modifications, and on the DNA this is seen as addition of methyl groups to certain C nucleotides in the promoter regions of the genes to be inactivated. In addition, the histone proteins that form molecular complexes with the DNA can be chemically altered, thereby changing the accessibility of the DNA to other proteins. With advancing differentiation, the genes required for the complex process of cell division are similarly blocked for use. Thus, fully differentiated and specialized cells, such as brain and heart muscle cells, are left inherently with a limited ability to divide and can thus dedicate all of their time to their assigned function in the body. Another consequence of these epigenetic changes that come with differentiation is that a cell differentiated in one direction cannot simply change into another differentiated cell type: the repertoire of genes that other cell types have available has been made inaccessible. Less differentiated cells, like multipotent stem cells, present in, for example, heart, pancreas and bone marrow, can use their genome somewhat more extensively and choose to become one of several possible subtypes of cells in the organ they are part of, while in addition they can still multiply to some extent. It will be evident that multipotent stem cells in an organ, such as the blood stem cells and mesenchymal stem cells in the bone marrow, are cells with even fewer epigenetic limitations. Finally, stem cells present in the early embryo are characteized by minimal DNA blocking.
EPIGENETICS IN STEM CELLS It has become clear that epigenetic changes, reflected in specific DNA methylation patterns, are often abnormal in embryonic stem cells that have been in culture in a laboratory for a long time. This implies that different embryonic stem cell lines are in this respect not necessarily identical, which probably explains why different cell lines often exhibit slightly different characteristics, for example in growth rate or the ease with which they differentiate in a certain direction. One reason for this is that during each cell division there is a chance of introducing an abnormality into the DNA being replicated which would therefore also be present in the two daughter cells. This way cells can accidentally acquire additional features, like mutations or abnormally methylated genes, which provide them with a growth advantage. Since rapidly proliferating cells tend to dominate and overgrow more slowly proliferating cells, they are gradually selected out during culture at the expense of cells with the original characteristics. In this way, after multiple passages in culture, when only the very distant relatives of the original cells are present, slightly different cell lines may evolve.
56
3. WHAT ARE STEM CELLS?
3.2
WHAT’S SO STRANGE ABOUT PLANARIA?
The regenerative capability of human tissues and organs is limited. A superficial wound will heal, but limbs or organs that are lost do not regrow. This is not true for all animal species. Less complex organisms often have remarkable regenerative capacities. Research into these species can teach us a great deal about the regenerative capability of tissues and pluripotency of cells. Planaria are kinds of small flatworms that have been investigated very extensively in the past. Planaria is a collective name for a variety of flatworms, including Schmidtea mediterranea and Dugesia japonica. Planaria are simple organisms, but they are composed of cells from three germ cell layers e just like mammals e namely the ectoderm, endoderm and mesoderm. They therefore have a nervous system and eyes (formed from ectoderm), muscles (from mesoderm) and a digestive tract (from endoderm). Planaria flatworms have been studied mostly because of their remarkable regenerative capacity. If a Planaria flatworm is cut into two parts, each half will grow to form a complete flatworm. If the head is separated from the body, or the body is cut into a number of pieces, each piece will form a new worm with a head and tail, each animal being genetically identical to the original animal e they are clones of the original animal. What is most interesting is that a piece of Planaria without a head will form a new head, but not an extra tail; Planaria without a tail will form a tail and no head. The cells that form new structures apparently also have positional information, know where they are in the Planaria’s body and somehow know exactly what to make. Planaria’s regenerative success is caused by a special group of cells with a large nucleus and little cytoplasm, called neoblasts. About 25e30% of Planaria cells are neoblasts. Little is known about these cells; it is still not clear whether they are formed from a self-renewing stem cell population or from specialized cells that de-differentiate. Neoblasts are probably totipotent, although it cannot be excluded that the neoblast population is intrinsically heterogeneous and that different neoblasts exist for various cell types. In intact Planaria, cell division takes place continuously, and new cells e formed from neoblasts e replace old cells that have served their function. If a Planaria is damaged, neoblasts migrate to the site of damage. Once there, they divide to increase the local number of neoblasts. These cells then differentiate into the required cell types and replace the damaged tissue. Exactly which signals make the neoblasts decide to
EPIGENETICS IN STEM CELLS
3.2
WHAT’S SO STRANGE ABOUT P L A N A R I A ? ( co nt ’d)
migrate to the wound, while preventing them from differentiating into different cell types within the organism, and how they obtain the exact positional information, are questions that remain unanswered. A better understanding of these mysteries may contribute to our knowledge of how human stem cells work.
If a Planaria flatworm is cut into several pieces, every part can reform (or regenerate) the missing piece so that within a few days each individual part has grown out to become a new flatworm.
57