Stem cells

The therapeutic application of stem cells has long been a dream of medical sciences, but recent discoveries and technical advances have brought this dream somewhat closer to being a reality. Stem cells are usually defined as undifferentiated cells capable of self-renewal that can differentiate into more than one specialized cell type.

Such cells are often classified on the basis of their original source as either embryonic or adult stem cells. As the name suggests, embryonic stem cells are derived from the early embryo, whereas adult stem cells are present in various tissues of the adult species. Much of the earlier work on embryonic stem cells was conducted using mouse embryos. Human embryonic stem cells were first isolated and cultured in the laboratory in 1998. Research on adult stem cells spans some four decades, with the discovery during the 1960s of haematopoietic stem cells in the bone marrow (Chapter 10). However, the exact distribution profile, role and ability to manipulate adult stem cells (particularly those outside of the bone marrow) are subjects of intense current research, and for which more questions remain than are answered.

Embryonic stem cells are derived from pre-implant-stage human embryos, usually at the blastocyst stage (the blastocyst is a thin-walled hollow structure containing a cluster of cells, known as the inner cell mass, from which the embryo arises). These embryos are invariably ones initially generated as part of in vitro fertilization procedures but which are destined to be discarded, either due to poor quality or because they are in excess to requirement. There are an estimated 400 000 in vitro fertilization-produced embryos in frozen storage in the USA alone, of which some 2.8 per cent are likely to be discarded.

Culture of human embryonic stem cells starts with the recovery of the blastocyst's inner cell mass (Figure 14.17). One common recovery procedure is termed 'immunosurgery'. The process

Culture on feeder cell layer Recovery of inner cell mass

Figure 14.17 Overview of the generation and culture of human embryonic stem cells. IVF: in vitro fertilization. Refer to text for further details entails the initial treatment of blastocysts with pronase (a cocktail of proteolytic enzymes), which effectively degrades the outer protective membrane known as the 'zona pellucida'. The blastocysts are next treated with anti-human whole serum antibody and guinea pig complement, which triggers complement-mediated lysis of the blastocyst outer cell layers (the trophoblast), allowing recovery of the inner cell mass. The latter cells are then cultured under defined conditions in order to allow them to multiply while remaining undifferentiated.

In addition to cell culture media, the culture vessels often contain a layer of 'feeder' cells (e.g. mouse fibroblasts), irradiated in order to prevent their growth and division. These feeder cells can serve two functions: (a) to provide a suitable substratum with which the embryonic stem cells can interact, aiding in their growth and division; (b) feeder cells can release often ill-defined nutrients into the medium, which can again support stem cell growth. The presence of a feeder cell layer would represent a complication in the downstream processing of stem cells for therapeutic use, and could represent a potential source of pathogenic contaminants. More recently, culture systems have been developed in which the feeder cell layer is replaced by fibronectin (a glycoprotein found on the cell surface) or matrigel (a protein-rich membrane extract from a mouse sarcoma cell line). Substantial research work remains ongoing in order to identify an optimal cell culture medium composition that will facilitate strong cell growth while remaining in an undifferentiated state. Basic animal cell culture media are often supplemented with serum as a nutrient source (Chapter 5). It is known that the addition of the cytokine LIF can sustain mouse embryonic stem cells in the undifferentiated state, but LIF alone cannot achieve this in the context of human embryonic stem cells. Research, therefore, continues with a view to optimize culture media composition for such human cell lines.

Whereas the culture of human embryonic stem cell lines requires the maintenance of cells in an undifferentiated state, the application of such cells in regenerative medicine requires the subsequent controlled differentiation of such cells to generate a specific desired cell type (e.g. a specific neuron type to treat a specific neurodegenerative disease, etc.). The process by which any stem cell differentiates naturally to form a specific cell is hugely complex and understood only in outline and only for a few cell types. Differentiation is dependent upon several concerted signals from effector molecules such as cytokines. A major challenge, therefore, is to gain a more complete understanding of how differentiation into specific cell types is driven and controlled. Only with such knowledge will come the ability to grow specific cells (and ultimately tissue/organ types) from stem cells for the purposes of regenerative medicine.

Although only in its infancy, some progress has been reported in elucidating details of selected directed differentiation pathways, initially in the context of mouse embryonic stem cells, but latterly also in the context of human embryonic stem cells (Figure 14.18). This progress has largely been the result of empirical studies and is largely achieved in one or more of three ways: (a) manipulation of culture media composition; (b) alteration of the surface characteristics of the matrix on which the cells are grown (e.g. adhesive feeder cells or specific protein-based matrices); (c) via introduction of specific regulatory genes into the stem cells themselves.

One example of a relatively recently elucidated pathway that directs differentiation of dopamin-ergic neurons is outlined in Figure 14.19. The ability to generate dopaminergic-like neurons represents a significant milestone in the attempt to apply regenerative medicine to the treatment of Parkinson's disease. This neurodegenerative condition, which effects some 2 per cent of adults over the age of 65, is triggered by the death of this cell type in the brain. Parkinson's disease, therefore, is likely to be one of the first clinical targets in the development of regenerative medicines.

Figure 14.18 Some cell types reported to have been produced via in vitro directed differentiation from either mouse or human embryonic stem cells. Potential uses for such cell types in regenerative medicines are listed in italics
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