Stem Cells
Figure 2: Combinatorial cell culture
Combicult™ is a high throughput platform for the
rapid identification of stem cell differentiation protocols. Stem cells on beads are exposed to multiple combinations of
media, containing active agents such as growth factors or small molecules, using a split- pool technique. The optimal combinations for effective
differentiation can be deduced rapidly and cost-effectively
differentiating into the three neural lineages of neurons, astrocytes and oligodendrocytes14 (Figure 1). Adult stem cells typically also have limited in vitro self-renewal capacity, although there are some exceptions, for example infinitely self-renewing neural stem cells have been isolated from foetal and adult brain15. Adult stem cells can be isolated from many adult and foetal tis- sues, eg haemopoietic, neural, mesenchymal and muscle2. Additionally, in some cases stable prolif- erating adult stem cells can be generated from pluripotent stem cells in vitro15,16.
Many factors have to be considered when devel- oping methods to culture and differentiate stem cells. The ability of stem cells to differentiate to multiple mature cells can be problematic in terms of obtaining high yield, pure populations of a par- ticular cell type. Stem cell differentiation typically requires serial cell culture steps with sequential
addition of particular combinations of growth and patterning factors, essentially mimicking processes that occur in vivo during development17. The microenvironment in which cells are cultured also needs to be optimised, as the extracellular matrix (ECM) substrate and spatial configuration of stem cells can have an enormous effect on their fate18. Testing a significant number of these variables is very labour intensive and time-consuming, limiting the development of optimised methods. Efficient identification of optimal stem cell differentiation protocols would greatly accelerate the widespread use of stem cells in industrial applications. Below we will describe some of the strategies for directing stem cell differentiation and the novel technologies which are driving increased understanding of stem cell biology and leading to improved methods for their differentiation.
Stem cell differentiation strategies 1. Soluble factors
The addition of growth factors or small molecules that target particular signalling pathways is one of the principal methods researchers use in attempt- ing to direct the differentiation of stem cells to a particular cell type. Selection of these factors is typ- ically based on what is known of lineage develop- ment during embryogenesis or in the adult during tissue repair. Different combinations of factors are typically added in a sequential manner, particular- ly for the differentiation of pluripotent stem cells, reflecting progressive lineage commitment (Figure 1). For example, the differentiation of hES cells to pancreatic cells requires a series of four different culture medium, over 36 days, which first induce stem cells to commit to definitive endoderm, then to pancreatic endoderm, to pancreatic endocrine/ exocrine cells and finally to more mature islet cells. Each medium contains a combination of growth factors and/or small
molecules19.To date, the development of such complicated protocols has been carried out empirically. However, recently high throughput approaches have been developed to accelerate protocol discovery.
The temporal, sequential nature of stem cell dif- ferentiation lends itself to a combinatorial approach to protocol discovery. Plasticell has developed a high throughput platform that uses combinatorial cell culture (Combicult™) technol- ogy to screen tens of thousands of protocols in one experiment20. Combicult™ combines miniaturisa- tion of cell culture on microcarriers, a pooling/splitting protocol and a unique tagging system to allow multiplexing of experiments. Stem cells grown on microcarrier beads are shuffled
74 Drug Discovery World Winter 2011/12
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