The Science of Stem Cells

1

What is a Stem Cell?

In the popular media and even in some medical circles, stem cells are presented as miracle cells that can do anything. When administered to a patient with some serious disease they will rebuild the damaged tissues and make the patient young again. Alas, in reality there are no such cells. However, there are cells that exhibit stem cell behavior and the future of regenerative medicine will undoubtedly be built on a good scientific understanding of their properties. In this chapter these properties are briefly outlined, and in the remainder of the book each of them will be underpinned by an explanation of the relevant areas of science and technology.

A list of characteristics of stem cell behavior that is generally agreed upon is the following:

Stem cells reproduce themselves.

Stem cells generate progeny destined to differentiate into functional cell types.

Stem cells persist for a long time.

Stem cell behavior is regulated by the immediate environment (the niche).

This is shown diagrammatically in Figure 1.1. The first two items on the list indicate the key abilities of self‐renewal and of generation of differentiated progeny. As will be explained below, these abilities may be shown at a cell population level rather than by every single stem cell at every one of its divisions. Also, the second item indicates destined to differentiate meaning that cell division may continue for a while before differentiation, but not indefinitely. Cells derived from stem cells that proliferate for a limited number of cycles are called progenitor cells or transit amplifying cells. The third item on the list means that if the stem cell population is one of those that exists in tissue culture then it should be capable of indefinite growth, while if it is part of an organism it should be very long lasting, normally persisting for the whole life of the organism. The fourth characteristic indicates that all stem cells exist in a specific micro‐environment that controls their program of division and differentiation. This may seem at first sight only to apply to stem cells within the body and not to those grown in vitro, but in order to get them to grow, the cells in vitro are always provided with specialized medium ingredients that, in effect, mimic the components normally provided in the niche.

Figure 1.1 A consensus diagram showing stem cell behavior.

(Modified from Slack, J.M.W. (2013). Essential Developmental Biology, 3rd edn. Reproduced with the permission of John Wiley and Sons.)

This fourfold definition involves not just intrinsic properties of stem cells, but also properties that depend on aspects of their environment such as the lifespan of the animal, the nature of the niche, or the composition of the culture medium. This emphasizes the fact that the goal of stem cell biology is understanding the behavior and not just the intrinsic nature of stem cells. To achieve this, the characteristics of the stem cell environment are just as important as the properties of the stem cells themselves. Moreover, understanding stem cell behavior means understanding various aspects of cell and developmental biology which are not always familiar to workers in stem cell laboratories.

The above definition is of value in indicating the special characteristics of stem cell behavior, but is also helpful in indicating what is not stem cell behavior. For example, most of the cells in the body that are dividing are not stem cells. In particular cells in the embryo that differentiate after a certain period of time, such as the earliest cells formed by division of the fertilized egg, are not stem cells. Nor are differentiated cells that divide during postnatal life to generate more of themselves, such as hepatocytes or tissue‐resident macrophages. A common term found in the literature is stem/progenitor cell. This is a singularly unhelpful designation as it conflates two entirely different cell behaviors. Progenitor cells are precisely those that differentiate into functional cell types after a finite period of multiplication. They include the transit amplifying cells that arise from stem cells (Figure 1.1) and also cells of the embryo and of the growing individual that are destined to differentiate after a certain time.

Real stem cells comprise two fundamentally different types: pluripotent stem cells that exist only in vitro, and tissue‐specific stem cells that exist in vivo in the postnatal organism. Pluripotent stem cells comprise embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). There are various subdivisions that will be considered later, but the essential features of these cells are first that they can be propagated without limit in vitro, and second that, under appropriate culture conditions, they are able to give rise to a wide variety of cell types, perhaps all the cell types in the normal organism except for the trophectoderm of the placenta. By contrast, tissue‐specific stem cells exist within the body and generate progeny to repopulate the tissue in question. Well‐studied tissue‐specific stem cells include those of the hematopoietic (blood‐forming) system, the epidermis, the intestinal epithelium and the spermatogonia of the testis. Under normal circumstances, tissue‐specific stem cells do not produce cells characteristic of other tissue types. There are also some well‐characterized stem cells that do not undergo continuous division, but seem to be kept in reserve to deal with tissue regeneration when required. A good example is the muscle satellite cells, which are normally quiescent but are able to be mobilized to divide and fuse to form new myofibers following injury. This type of stem cell behavior is sometimes called facultative.

Many criteria for identifying stem cells have been proposed and used. These are briefly listed here and the concepts and technologies will be developed in later chapters of the book.

Stem Cell Markers

Very often a cell is said to be a stem cell because it expresses one or more gene products associated with stem cells. However, there is no molecular marker that identifies all stem cells and excludes all non‐stem cells. Those components required for general cell metabolism and cell division are certainly found in all stem cells, but they are also found in many other cell populations as well.

Pluripotent stem cells (ESC and iPSC) express an important network of transcription factors which are necessary for maintenance of the pluripotent state (see Chapter 6). Transcription factors are the class of proteins that control the expression of specific genes. A key member of the pluripotency group is the POU‐domain transcription factor OCT4 (also known as OCT3 and POU5F1). The presence of OCT4 is certainly necessary for the properties of pluripotent stem cells. However it is not expressed in any type of tissue‐specific stem cells except at a low level in spermatogonia.

A component that might be expected to be found in all stem cells is the telomerase complex. At the end of each chromosome is a structure called the telomere, made up in vertebrate animals of many repeats of the simple sequence TTAGGG. Because of the nature of DNA replication, the double helix cannot be copied right up to the end, so a part of the telomere is lost in each cell cycle. After enough cycles, the erosion of chromosome ends activates the system which senses DNA double‐stranded breaks and causes death of the cell. This process is an important reason for the limited survival time of most primary tissue culture cell lines, which undergo senescence after a certain number of population doublings in vitro. Obviously there must be a mechanism for repairing telomeres in vivo, and this is provided by the telomerase complex, of which the most important components are an RNA‐dependent DNA polymerase called TERT, and an RNA called TERC which contains the template CCCTAA for the telomere sequence. High levels of telomerase are found in germ cells, ensuring the survival of full length chromosomes for the next generation. Telomerase is also upregulated in permanent (transformed) tissue culture cell lines and in most cancers. However most types of somatic cell have little or no telomerase. Tissue‐specific stem cells do contain some telomerase; generally enough to maintain cell division for a normal lifetime, but not enough to fully reverse the erosion of the telomeres. In situations such as repeated transplantation of hematopoietic stem cells from one mouse to another, there is an upper limit to the number of possible transplants and this is determined at least partly by telomere erosion. The presence of telomerase can be considered to be a stem cell marker, although it is also found in permanent tissue culture lines, early embryos and most cancers.

In human or animal tissues, various markers have been advanced as characteristic of all stem cells. For example the cell surface glycoprotein CD34 is found on human hematopoietic stem cells (HSCs) and can be used to enrich them from bone marrow by fluorescence‐activated cell sorting (FACS). However it is also found on other cell types, such as capillary endothelial cells, and it is unclear whether it is actually necessary for the stem cell behavior of the HSC. In fact, since it is not found on mouse HSC, which are generally similar in behavior to human HSC, it is probable that it is not necessary. CD34 is not found on human embryonic stem cells or on most epithelial stem cell types, indicating that it is not a generic stem cell marker. A molecular marker which is known to be required for stem cell function is LGR5. This is an accessory receptor for the Wnt family of signaling molecules (see Chapter 7) and is found on stem cells in the intestine, hair follicle, mammary gland and stomach. These types of stem cell all depend on Wnt signaling from their environment for continued cell division, so the presence of the LGR5 is really necessary. However it is not found on other types of stem cell, so is also not a universal marker.

An interesting type of marker is that offered by dye exclusion, in particular exclusion of the Hoechst 33342 dye. This is a bisbenzimide dye, excited by UV light to emit a blue fluorescence. It is widely used as a DNA‐binding reagent, but it is also actively pumped out of some cell types. If a subgroup of cells has lost more dye than the rest of the population, then it appears in flow cytometry as a cluster of cells showing less blue fluorescence than average. This is called a side population. The side population is enriched for stem cells in some situations, especially in murine bone marrow where it provides a similar degree of enrichment of hematopoietic stem cells to FACS using a panel of cell surface markers (Figure 1.2). The dye exclusion property is due to the activity of cell membrane transporter molecules including the P‐glycoprotein (MDR1) and transporters of the ABC class. Dye exclusion is indicative of an increased capacity for export of all hydrophobic small molecules, many of which are toxic to cells. Although useful to the investigator, it is unlikely that this capacity is really important for stem cell behavior. For example, mouse embryonic stem cells show dye exclusion while human embryonic stem cells do not.

Figure 1.2 Flow cytometry plots showing a side population of cells active in Hoechst dye exclusion. (a) Whole mouse bone marrow, the boxed region is the side population. (b) Side population cells refractionated with regard to differentiated lineage markers, absent from stem cells, and Sca‐1, a cell surface marker present on stem cells.

(From: Goodell, M.A., Brose, K., Paradis, G., Conner, A.S. and Mulligan, R.C. (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. Journal of Experimental Medicine 183, 1797–1806. Reproduced with the permission of The Rockefeller University.)

In summary, there is no single gene product which is found in all stem cells and not in any non‐stem cell. Many so‐called stem cell markers are probably not necessary for stem cell behavior. Of those gene products which are necessary for stem cell behavior, some, such as the cell division machinery and telomerase, are found in stem cells and in some non‐stem cells. Others, such as OCT4 or LGR5, are found in some, but not all, types of stem cell.

Label‐Retention

When a cell population is exposed to a DNA precursor, such as the nucleoside bromodeoxyuridine (BrdU), which is metabolized by cells in the same way as thymidine, all cells undergoing DNA synthesis will incorporate it into their DNA and so become labeled. The BrdU in the cell nuclei can be detected by immunostaining. After the BrdU supply is withdrawn, so long as cell division is continuing, then the level of BrdU in the DNA will halve with every subsequent S phase and become undetectable to immunostaining after about six divisions. If a cell divides slower than average, it will retain detectable BrdU for longer. This label‐retaining behavior is often considered to be a characteristic of stem cells. In Figure 1.C.1 is shown an image of a hematopoietic stem cell (HSC) visualized with an antibody to the cell surface marker CD150. It retains a DNA precursor (EdU) label from a pulse given 30 days previously. Likewise, muscle satellite cells, that enable muscle regeneration following damage, are usually in a quiescent state. This relatively quiescent behavior is considered necessary to maintain regenerative function of some types of stem cell over a lifetime. If the mechanisms of quiescence are disturbed in mice by knocking out key components, then hematopoietic stem cells or muscle satellite cell populations have been shown to become exhausted during the lifetime of the animal, because they are dividing too much. Relative quiescence also serves to protect the stem cells against the oxidative damage which results from continuous growth with its associated oxidative metabolism.

Slow division is the cause of label retention in stem cells, but it must be remembered that not all label retention is due to stem cell behavior. In particular differentiation to a completely non‐dividing (post‐mitotic) cell type leads to permanent label retention. This property has been used especially to establish the differentiation time of neurons in embryonic development, and the final mitosis is often referred to as the cell birthday. Moreover, label retention is by no means universal among stem cells. For example, it is not shown by intestinal or epidermal stem cells. It is also, of course, not shown by the pluripotent stem cells (ES or iPS cells) which undergo rapid division in culture.

The Niche

The concept of a stem cell niche arose in the 1970s to explain the fact that the spleen colony‐forming cells from the bone marrow had a lesser differentiation potency than hematopoietic stem cells in vivo (see Chapter 10). The idea is that stem cells require continuous exposure to signals from surrounding cells in order to maintain their stem cell behavior. This was first proved experimentally using the fruit fly Drosophila. In the Drosophila ovary there are female germ cells which lie in contact with somatic cells called cap cells. These secrete a TGFβ-like molecule called Decapentaplegic (Dpp). Dpp maintains the stem cells in mitosis. But as they divide, some of the stem cell progeny become displaced from contact with the cap cells, and are then exposed to less of the Dpp. This fall in Dpp lifts a repression on the oocyte maturation process and enables the daughter cell to differentiate to a cystoblast. This then undergoes a fixed differentiation program, dividing four times to generate a post-mitotic complex of one oocyte and 15 supporting nurse cells. This situation illustrates the behavior of a niche very nicely. The stem cells continue to divide so long as they are in contact with the niche, and they differentiate when they are no longer in contact. If a stem cell is removed experimentally, its position may be taken by a progeny cell which would normally have differentiated, but because of its renewed occupancy of the niche it remains a dividing stem cell.

Figure 1.3 The stem cell niche in the Drosophila ovary. Female germ cell stem cells require continued contact with cap cells to remain stem cells. Once they lose contact with cap cells they differentiate into a cyst of one oocyte and 15 nurse cells.

(Slack, J.M.W. (2009) Essential Developmental Biology, 2nd edn. Reproduced with the permission of John Wiley and Sons.)

Probably all the stem cells types in the mammalian body exist within specific niches like this which control their behavior. For example the intestinal stem cells lie adjacent to Paneth cells which supply WNT, and spermatogonial stem cells lie adjacent to Sertoli cells that supply them with glial derived neurotrophic factor (GDNF). In both cases the signaling molecules are needed to maintain the stem cells in mitosis, and removal from the niche brings an end to cell division unless the factors are provided experimentally. In the bone marrow, there has been controversy about the exact nature of the niche, but hematopoietic stem cells are often found adjacent to blood vessels, as shown in Figure 1.C.1.

Asymmetric Division and Differentiated Progeny

It is often thought that all stem cells must undergo asymmetric divisions, with one daughter being a stem cell and the other destined to differentiate. This does sometimes occur, but it is also possible for stem cells to have a less rigid program of cell division with some divisions producing two stem cells, some two progenitor cells, and some producing one of each. Statistically a steady state requires that the stem cell number remains constant, although there may be occasions where it needs expanding, such as during normal growth of the organism or following injury. In the intestine for example it has been shown by cell labeling and by direct visualization that symmetric divisions predominate (see Chapter 10).

By definition, stem cells must produce differentiated progeny, but how many differentiated cell types do they actually produce? The answer is very variable and depends on the tissue concerned. In the intestine, stem cells produce absorptive, goblet, tuft and Paneth cells, together with several types of enteroendocrine cells. In the bone marrow, the hematopoietic stem cells produce all the cell types of the blood and immune system. At the other end of the scale, the spermatogonia of the testis produce only sperm. Epidermal stem cells are often said to produce only keratinocytes, but they can also form a type of neuroendocrine cell called the Merkel cell, responsible for touch sensitivity. The examples of both the intestine and the epidermis indicate that neuroendocrine cells can arise from epithelial stem cells quite distinct from the central or peripheral nervous systems, but they are not indicative of a wider potency enabling other tissue types to be