Biological Considerations

Stem Cells

It is now thought that essentially all tissues contain stem cell populations that can produce cellular progeny that differentiate into mature tissue phenotypes. The maturational process includes two branches: the “commitment “branch, in which pluripotent stem cells produce daughter cells with restricted genetic potentials appropriate for a single set of cell activities (unipotent), and the “differentiation” branch, in which sets of genes are activated and/or altered in their levels of expression. The following sections discuss how these major tissue subdivisions work together to generate or repair tissues in order to provide tissue functions.

Stem Cells and the “Niche” Hypothesis

Pluripotent stem cells are cells that are capable of producing daughter cells with more than one fate; they can self-replicate, and they have the ability to produce daughter cells identical to the parent. Totipotent stem cells are cells that can generate all the cell types of the organism.

Determined stem cells are cells in which the genetic potential is restricted to a subset of possible fates; they can produce some, but not all, of the cell types in the organism. Determined stem cells of the skin can produce all the cell types in the skin but not those of the heart. Similarly, determined stem cells of the liver can produce all liver cell types but not brain. The lay press often refers to determined stem cells as “adult stem cells,” which is a misnomer because determined stem cells are present in fetal and adult tissue. The determined stem cells give rise to unipotent progenitors, also called committed progenitors, with genetic potential restricted to only one fate. These unipotent progenitors rapidly proliferate into large numbers of cells that then differentiate to mature cells. The stem cells and the unipotent progenitors are the normal counterparts to tumor cells and to immortalized cell lines. Determined stem cells identified to date are small in size (typical diameters of 6–10 mm), have a high nucleus to cytoplasmic ratio (blast-like cells), and express certain early genes (e.g., alpha-fetoprotein) and antigens (e.g., CD34, CD117). They have chromatin that binds particular dyes at levels lower than that of the chromatin in mature cells, enabling them to be isolated as “side-pocket” cells using flow cytometric technologies. Stem cells express an enzyme, telomerase, that maintains the telomeres of their chromosomes at constant length, a factor in their ability to divide indefinitely in vivo and ex vivo. Multiple parameters must be used to permit isolation and purification of any determined stem cell type, since there is no one parameter (antigen, size, cell density) sufficient to define any determined stem cells. Furthermore, they appear to grow very slowly in vivo and may commit to growth and differentiation in a stochastic manner. A first-order rate constant for hemopoietic stem cells is about one day and their cycling times have been measured by means of time-lapse videography. They commit to differentiation in culture. The first and second doubling take about 60 hours, and then the cycling rate speeds up to about 24 hours cycling time. By the fifth and sixth doubling, they are dividing at a maximal rate of 12 to 14 hours doubling time.

What evidence is there that stem cells exist?

Lethally irradiated mice that would otherwise die from complete hematopoietic failure can be rescued with as few as 20 selected stem cells. These animals reconstitute the multiple lineages of haematopoiesis as predicted by the stem cell model. In sub lethally irradiated animals, genetically marked mesenchymal stem cells found in bone marrow will give rise to cells in multiple organs over a long time period. These investigations and many others have established conclusively the presence of stem cells, their multilineage potential, and their ability to persist over long periods of time in vivo.

Stem Cell Niches

The field of stem cell niches is rapidly expanding due to its importance in regulating stem cell fate. The goal is to define and understand the local microenvironment of the stem cell compartment. The field is still new but already has yielded generalizations that are proving to be useful guides for defining ex vivo expansion conditions for the cells:

• Stem cells do not have the enzymatic machinery to generate all their lipid derivatives from single lipid sources and so require complex mixtures of lipids for survival and functioning.

• Calcium concentrations are quite critical in defining whether stem cells will expand or undergo differentiation. The mechanisms underlying the phenomenology are poorly understood.

• Specific trace elements, such as copper, can cause more rapid differentiation of some determined stem cell types. It is unknown whether this applies to all stem cells, and the mechanism(s) is not known.

• Specific mixtures of hormones and growth factors are required, with the most common requirements being insulin and transferrin/Fe. Addition of other factors can result in expansion of committed progenitors and/or lineage restriction of the stem cells toward specific fates.

• The matrix chemistry of known stem cell compartments consists of age-specific and cell-type-specific cell adhesion molecules, laminins, embryonic collagens (e.g., type III and IV collagen), hyaluronans, and certain embryonic/fetal proteoglycans. With maturation of the stem cells toward specific cell fates, the matrix chemistry changes in a gradient fashion toward one typical for the mature cells. Although the matrix chemistry of the mature cells is unique for each cell type, a general pattern is the inclusion of adult-specific cell adhesion molecules, various fibrillar collagens (e.g., type I, II collagen), fibronectins, and adult-specific proteoglycans. A major variant is that for skin and neuronal cells, in which mature cells lose expression of collagens, fibronectins, and laminin. Additionally, the matrix chemistry of these cell types is dominated by CAMs and proteoglycans.

• Stem cells are dependent on signals from age- and tissue-specific stroma. The signals from the stroma are only partially defined but include signals such as Leukaemia Inhibitory Factor (LIF), various fibroblast growth factors or FGFs, and various interleukins (e.g., IL 8, IL11).

• The most poorly understood of all the signals defining the stem cell niche(s) are those from feedback loops, which are initiated from mature cells, and where these signals inhibit stem cell proliferation. Implicit evidence for feedback loops is that cell expansion ex vivo requires separation between cells capable of cell division (the diploid subpopulations) and the mature nonproliferating cells.

Which tissues have stem cells?

For decades it was assumed that stem cell compartments exist only in the rapidly proliferating tissues such as skin, bone marrow, and intestine. Now, there is increasing evidence that essentially all tissues have stem cell compartments, even the central nervous system. There are now numerous reports of the isolation and characterization of tissue-specific stem cells, and they are actively being investigated as a potential cell source for tissue engineering.

The Roles of Stem Cells

The stem cell compartment of a tissue is the fundamental source of cells for turnover and regenerative processes. Stem cell commitment initiates cell replacement and genesis of the tissue, resulting in tissue repair and maintenance of tissue functions. Stem cell depletion due to disease or toxic influences (e.g., drugs) eventually leads to partial or complete loss of organ function. Mutational events affecting the stem cells can result in tumors for which both altered and normal stem cells are actively present. Thus, tumors are now considered transformed stem cells, an idea originally proposed by Van Potter, Sell, and Pierce, and now confirmed by current stem cell biologists.

The Maturational Lineages of Tissues

All stem cells are pluripotent, giving rise to multiple, distinct lineages of daughter cells that differentiate, stepwise, into all of the mature cells of the tissue. A general model for the production of mature cells arising from tissue-specific stem cells is shown in Figure 6.9. Determined stem cells (pluripotent) replicate slowly in vivo, with rates influenced by various systemic signals. Their immediate descendants are committed progenitors (unipotent) capable of rapid proliferation and shown in some tissues (e.g., skin) to be the acute responders to mild to moderate regenerative stimuli. The unipotent progenitors mature in a stepwise fashion through intermediate stages into fully mature cells. Characteristically, various tissue-specific functions are expressed in cells throughout the maturational lineage and in a lineage-dependent fashion. One can generalize about these gradual phenotypic changes by categorizing the functions as “early,” “intermediate,” and “late” taskings. In some cases, a specific gene is expressed uniquely only at a specific stage. In others, there are isoforms of genes that are expressed in a pattern along the maturational lineage, while in yet others there are changes in the levels of expression of the gene. Finally, the cells progress to senescence, a phenomena that typifys aging cells. The maturation of the cells is dictated in part by mechanisms inherent in the cells (e.g., changes in the chromatin)

FIGURE 6.9 Model for cell production in prolific tissues. This model was derived from decades-long research in haematology. The columns represent increasingly differentiated cells, while the rows indicate the cellular fate processes (Figure 16.4) and other events that cells undergo at different states of differentiation. (td denotes doubling time.)

and in others by matrix and/or soluble signals in their microenvironment. The microenvironment can include signalling affecting growth, differentiation, or apoptosis. For example, some growth factors are survival factors with antiapoptotic effects.

Examples of Stem Cell–Fed Maturational Lineages

The stem cell models best characterized are the hemopoietic stem cells, the intestinal stem cells, and the skin stem cells. In addition, bone marrow–derived mesenchymal stem cells and stem cells of the liver are being increasingly investigated. These systems are described further in the following sections.