Biomedical Engineering Reference
In-Depth Information
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 blasto-
cysts 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 defi ned 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 fi broblasts), 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-defi ned 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 fi bronectin (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 subse-
quent controlled differentiation of such cells to generate a specifi c desired cell type (e.g. a specifi c
neuron type to treat a specifi c neurodegenerative disease, etc.). The process by which any stem cell
differentiates naturally to form a specifi c 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 specifi c cell types is driven and controlled. Only with
such knowledge will come the ability to grow specifi c 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 lat-
terly 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) ma-
nipulation 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 specifi c protein-based matrices); (c) via
introduction of specifi c 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 repre-
sents a signifi cant milestone in the attempt to apply regenerative medicine to the treatment of Par-
kinson'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 fi rst clinical targets in the development of regenerative medicines.
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