ISCI 2
Despite a steady increase in the numbers of researchers over the last decade since human embryonic stem cells were isolated several practical difficulties exist in the growing and maintenance of the cell lines. Most notable amongst these are the tendency for human embryonic stem cells to acquire genetic changes during long term culture and complex substrate and media requirements for the maintenance of the stem cell state.
Improving Growth Conditions
When first derived hES cultures essentially adopted the same culture conditions as the original mouse ES derivations i.e. the use of primary mouse feeder cells with a serum or serum-like based basal media.
More recently several signalling pathways have been identified as playing a significant role in hES cell self-renewal. In particular FGF, TGF-beta, BMP, WNT have all been proposed to modulate the behaviour of ES cells in culture. Functional studies have indicated that in particular high levels of FGF signalling coupled with suppression of BMP signalling by the use of enhanced TGF-beta signalling promote the self-renewal of hES cells in vitro. It is the presence and apparent activity of these pathways that has lead to a number of rationally designed growth media for hES culture.
However a number of points need to be addressed:
- Understandably Laboratories developing media can only test the media on the hES isolates they have available. This means that in practice any given media has only been tested on a limited number of cell lines.
- Given the increasing numbers of cell lines being derived by different methods and for different purposes it is imperative that researchers have available to them media which are known to be robust supporters of the stem cell state in a wide range of cell lines.
- Researchers studying signalling pathways and control of hES self-renewal and differentiation will require fully defined media.
With these points in mind it was decided to test a selection of published media formulations for their ability to maintain a wide range of cell lines in a wide range of laboratories.
The study is divided into three parts:
Phase 1
Basic experimental design:
- 9 media were selected from the literature
- Labs to grow three cell lines in 9 media formulations (batches of three) for up to 10 passages
- RNA Expression, Flow Cytometry for a panel of antigens and karyotype assessed at the start and end
- Cell growth assessed throughout the time of the experiment
Summary of Phase One
Key project stages:
- Media formulations identified from literature
- Study design and media formulations (9) to be used finalized at the 3rd ISCI Workshop, The Jackson Laboratory, Maine. USA
- Media components sourced for distribution to participating laboratories
- Media testing begins in batches of 3 media + control
- Participating Laboratories informally liase on interim results.
- Data analysis
- Decision made on ‘short-list’ of media to be taken forward to phase 2
Participating Laboratories
The following laboratories are to carry forward the phase one study:
- Broad Institute, University of Southern California. USA- Martin Pera
- WiCell Research Institute, USA - Tenneille Ludwig
- Karolinska Institute, Sweden- Outi Hovatta
- Kyoto University, Japan – Norio Nakatsuji
Phase 2
Experimental Design
Key project stages
- Conformation on identity of testing laboratories
- Components to manufacture Short-listed media sourced and shipped to labs
- Test phase (40 passages, approximately 1 year)
- Data collection and analysis
- Report to consortium
Participating laboratories
To be decided
In addition to the two test phases described above a set experiments, coordinated by Peter Zandstra, University of Toronto, will seek to address what components common to the reported growth media are in fact critical hES cell self-renewal.
Genetic Stability
As part of the ongoing efforts started in ISCI-1 to characterize further human embryonic stem cell isolates a prospective study is being undertaken to catalogue the range of karyotypic changes that are seen in hES isolates worldwide. It is hoped that by following a large cohort of cell lines a more definitive picture of common karyotypic changes and minimal amplicons might be identified.
