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Fundamentals of the Stem Cell Debate

University of California Press

Chapter One

Peter J. Bryant and Philip H. Schwartz

WHAT ARE STEM CELLS?

Stem cells are undifferentiated cells found in the embryos and the later life stages of animals, including humans. They are recognized by their dualistic nature: they either can expand their numbers (self-renew) while remaining undifferentiated or can differentiate and contribute to the development or repair of tissues of the body. Some authors have added other criteria to the definition, including the ability to produce cells differentiating in different ways (multipotency); the ability of a single cell to proliferate into a population of similar cells (clone-forming ability); and the ability to keep dividing indefinitely (unlimited proliferative capacity)-the latter property distinguishing them from most other noncancerous cell types, which can undergo only a limited number of divisions. In most examples of stem cells only some of these properties have been demonstrated, and the term stem cell has been used fairly loosely. However, stem cells of many types are now being intensively studied by genetic and molecular methods, and biologists are developing more rigorous and convenient methods to identify them. They are recognized by their expression of certain genes, their production of characteristic proteins and antigens, and their responsiveness to certain growth factors.

In the best-analyzed examples of stem cells in experimental organisms, self-renewal is accomplished through conventional symmetric cell division (figure 1), whereas differentiation is controlled through a specialized mechanism called asymmetric cell division (ACD; figure 1). ACD results in the budding of a (usually) smaller cell from the larger stem cell (Potten 1997). Through this division the stem cell renews itself and can undergo more such divisions, while the other cell either begins to differentiate or undergoes a small number of additional divisions before the resulting cells differentiate.

When a cell begins the process of ACD, one set of specialized proteins accumulates on one side of the cell and another set accumulates on the other (figure 2). These proteins (and some messenger RNAs) are then included either in the stem cell or in the differentiating cell. Furthermore, experimental studies show that these localized molecules actually control whether the cell receiving them remains a stem cell or begins differentiating. The molecules are therefore called ACD determinants. Most of them have been identified through genetic studies of ACD during the development of the nervous system in the fruit fly Drosophila. In the absence of any one of the ACD determinants the asymmetry of division is disrupted, and this leads to abnormal cell proliferation and/or abnormal cell fates. Some of the ACD determinants control the localization of others, and the molecular interactions between them are under active study (Matsuzaki 2000).

Most of the proteins implicated in ACD in Drosophila have remarkably close mammalian and human counterparts (homologs), but there is only fragmentary evidence regarding the possible roles of these homologs in the control and division of mammalian stem cells. Much of the information comes from work on the formation of the nervous system in the mammalian embryo, where ACD has been demonstrated in the mouse (Shen et al. 2002) and ferret (Chenn and McConnell 1995). Preliminary studies have suggested that ACD during mammalian development is controlled by the homologs of some of the ACD determinants identified in Drosophila, including those named Numb, Numblike, Notch1 (Fang and Xu 2001; Justice and Jan 2002; Zhong et al. 1997; Zhong et al. 1996), and LGN (homolog of Drosophila Pins; Fuja et al. 2004; Mochizuki et al. 1996). In one of the most definitive studies, stem cells were isolated from the living embryonic mouse brain and cultured through a division cycle, and the resulting cell pairs were stained using antibodies against the Numb protein (Shen et al. 2002). The protein often accumulated in one of the two daughter cells, and this accumulation was correlated with the subsequent fates of the daughter cells. The Notch signaling pathway, identified genetically in Drosophila, also seems to be involved in ACD of satellite cells during mammalian muscle development (Conboy and Rando 2002).

The fate of stem cells as well as the way they divide appears to be a function of their microenvironment, which in many cases is provided by a specialized structure known as the stem cell niche. At least in the hematopoietic (blood cell-forming) system, the niche develops independently and the stem cells migrate to and colonize the niche (Schofield 1983). It has been suggested that the niche controls the phenotype of the stem cell, including whether it undergoes self-renewal or ACD. Evidence suggesting the existence of stem cell niches has also been obtained for the epidermis, intestinal epithelium, nervous system, and gonads (Fuchs, Tumbar, and Guasch 2004), as well as in developing muscles (Venters and Ordahl 2005). Furthermore, some of the soluble growth factors mediating interaction between niche and stem cells have been identified (Hauwel, Furon, and Gasque 2005).

EMBRYONIC STEM CELLS (ESCs)

In the mammalian embryo, following fertilization of the egg by a sperm, several cell divisions take place without any growth in total volume (figure 3), so the cells (now called blastomeres) get progressively smaller. They also rearrange to form a hollow sphere of cells (blastocyst) surrounding a fluid-filled cavity called the blastocoel. The cells of the blastocyst then segregate into an outer layer, called the trophectoderm, and an inner cell mass (ICM). The cells of the trophectoderm (trophoblasts) become the fetal contribution to the placenta, while the ICM contains the embryonic stem cells (ESCs) that give rise to the tissues of the fetus (figure 4).

Isolation

Human ESCs (hESCs) are usually obtained from the ICM of embryos produced by in vitro fertilization (IVF). In this procedure, eggs are harvested from a woman after she has been treated with follicular hormones to stimulate the ovaries. The eggs are fertilized either by combining them with sperm in a dish or by mechanically injecting the sperm into the egg (intracytoplasmic sperm injection). The latter technique has the advantage that every egg gets fertilized and that only one sperm enters each egg. The fertilized eggs are then incubated to allow them to develop into blastocysts. Then the trophectoderm is removed and the ICM is plated on to a "feeder layer" of mouse or human embryonic fibroblasts (Thomson et al. 1998), which is essential for the survival of the ICM (Cowan et al. 2004). The ICM then flattens into a compact colony of ESCs. ESC colonies are then mechanically dissociated and replated several times to give rise to stable cell lines.

Properties

Under certain conditions hESCs can divide indefinitely while undifferentiated, but under other conditions they can differentiate into virtually any cell type in the body (Amit et al. 2000; Bodnar et al. 2004; Cowan et al. 2004; Odorico, Kaufman, and Thomson 2001; Thomson et al. 1998). When undifferentiated hESCs are transplanted into an animal, they often form a type of tumor called a teratoma (Altaba, Sanchez, and Dahmane 2002), which is unusual in that it contains cells representing all three germ layers (Trounson 2004). Indeed, the ability of hESCs to form a teratoma after injection is the accepted criterion for identifying hESCs as such.

When cultured in the laboratory, hESCs grow as compact colonies and usually require the presence of "feeder cells" for their survival (figure 5). The feeder cells are typically mouse fibroblasts that have been treated with mitotic inhibitors to prevent their proliferation. But to make hESCs safe for use in human cell therapy, methods are being developed in which the human cells have no contact with animal cells. Human feeder cells can be effective (Amit et al. 2000). Another possibility is to first condition the culture medium by incubating it with feeder cells, then remove the feeder cells and use the conditioned medium, presumably containing appropriate growth factors, for culturing the stem cells (Carpenter et al. 2004; Rosler et al. 2004; Xu et al. 2001).

Human ESCs have specific requirements for nutrients, including "serum replacement medium." Serum is a necessary component for survival and/or differentiation of many cell types, but it invariably induces differentiation of hESCs, so it cannot be used to promote their survival and/or proliferation. This problem has been overcome by the use of serum replacement medium, which has many of the supportive properties of serum but lacks the tendency to cause differentiation. Another feature of hESCs is their inability to divide and/or survive in low-density culture. When they are dissociated into a single cell suspension, these cells have a very low survival rate. Colonies are therefore usually mechanically dissected into smaller colonies, rather than dissociated into single cells, for propagation.

Human ESCs in culture have a specific morphology, and they express characteristic surface antigens and nuclear transcription factors. The surface antigens include the stage-specific embryonic antigen SSEA-4 and the teratocarcinoma recognition antigens TRA-1-60 and TRA-1-81 (Carpenter et al. 2004). The transcription factors include the POU (pit-oct-unc)-domain transcription factor Octamer-4 (Oct-4), associated with the expression of particular elements of the embryonic genome (Thomson et al. 1998).

Differentiation

When undifferentiated hESC colonies are detached from the feeder layer and transferred into serum-containing medium, they form multicellular aggregates called embryoid bodies (EBs, figure 6), which can contain cell types representing all three germ layers of the body-endoderm, mesoderm, and ectoderm (figure 4). Many EBs tend to show cell types of only one or two germ layers, but in an unpredictable manner. Thus, with appropriate subculture conditions and physical removal of colonies showing specific morphologies, behaviors, or proteins, it is possible to establish cultures that are enriched for particular cell types or mixtures of cell types (figure 6; Carpenter et al. 2004). However, this cell behavior is unpredictable and the sorting is not completely effective. Many labs have therefore been trying to develop protocols for directly controlling the differentiation of hESCs.

Exogenous differentiating factors have been useful in favoring differentiation into specific derivatives: retinoic acid and nerve growth factor for neuronal differentiation (Schuldiner et al. 2001); basic fibroblast growth factor and platelet-derived growth factor for glial precursors (Brustle et al. 1999); 5-aza-2'-deoxycytidine for cardiomyocytes (Xu et al. 2002); bone morphogenetic protein-4 and transforming growth factor-beta for trophoblast cells (Carpenter, Rosler, and Rao 2003); sodium butyrate for hepatocytes (Rambhatla et al. 2003); and various cytokines for hematopoietic cells (Zhan et al. 2004). Differentiation into particular tissue types can also be elicited by overexpressing genes encoding transcription factors that function in cell commitment during normal development: MyoD1 for skeletal muscle (Dekel et al. 1992) and Nurr1 for dopamine neurons (Kim et al. 2002). However, these methods still usually give only enrichment rather than total induction, so additional sorting is often necessary. This has been done on the basis of lineage-specific gene expression: PS-NCAM and A2B5 as cell-surface markers for neural precursors (Carpenter et al. 2001), or hygromycin resistance driven by a myosin heavy chain promoter for cardiomyocytes (Klug et al. 1996) (figure 6).

Several groups (Brustle et al. 1999; Reubinoff et al. 2001; Tabar et al. 2005; Wernig et al. 2004) have produced neuronal precursors from either mouse or human ESCs and tested them by injection into the developing brain of newborn mouse or embryonic rat. The transplanted cells were incorporated into the host brain, migrated along appropriate tracks, differentiated into neurons in a region-specific manner, and made synaptic contacts with host neurons. In some cases the transplanted cells also gave rise to glia and astrocytes. This procedure has been shown to promote recovery in animal models of Parkinson's disease and spinal cord injury (Shufaro and Reubinoff 2004).

NEURAL CREST STEM CELLS

A peculiar and heterogeneous population of migratory precursor cells, called neural crest cells, originates during fetal development from the neural folds at the dorsal side of the neural tube. These cells migrate through the embryo to differentiate into a bewildering collection of derivatives, including most of the neurons, Schwann cells, and glia of the peripheral nervous system; most primary sensory neurons; some endocrine cells in the adrenal and thyroid glands; smooth muscle associated with the heart and great vessels; pigment cells of the skin and internal organs; and bone, cartilage, and connective tissue of the face and neck (Le Douarin and Dupin 2003). The migrating cells include multipotential neural crest stem cells, but the population becomes progressively restricted, and terminal differentiation usually ensues soon after the cells reach their targets (Baroffio, Dupin, and Le Douarin 1991). However, some studies show that neural crest-derived stem cells can still be identified in adult organs, including the central nervous system (Altman 1969; Doetsch et al. 1999; Eriksson et al. 1998; Gould et al. 1999; Johansson et al. 1999; Palmer, Takahashi, and Gage 1997; Reynolds, Tetzlaff, and Weiss 1992) and the hair follicle (Sieber-Blum et al. 2004). Some of the other reported examples of adult stem cells, described below, have not yet been adequately tested to see whether they might also have a neural crest origin. Neural crest-derived cells can be identified by the expression of the neural crest marker Sox-10 (Sieber-Blum et al. 2004).

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Excerpted from Fundamentals of the Stem Cell Debate by K. R. Monroe Copyright © 2008 by The Regents of the University of California. Excerpted by permission.
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