Building on the success of ISCI1, The International Stem Cell Forum approved funding for a second Initiative, ISCI2, that focussed upon comparing the performance of different media for the culture of hES cells, and assessing the types of enetic change that accumulate in hES cells upon prolonged passage.
The Scientific Management of the ISCI Program is provided by a Steering Committee, comprising;
- Peter W. Andrews (Chair) university of Sheffield, UK
- NissimBenvenisty – The Hebrew University of Jerusalem, Israel
- Barbara B. Knowles – A*STAR, Singapore
- Martin F. Pera – Stem Cells Australia and the University of Melbourne, Australia
- Janet Rossant – The Hospital for Sick Children, Toronto, Canada
- Glyn N Stacey – UK Stem Cell Bank, UK
- Steve K. W. Oh – Bioprocessing Technology Institute, Singapore
Contact the ISCI Team
Administration: Please email Nick Childs
Improving Growth Conditions
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 the robustness and general applicability of different media formulations to different hES cells needed to be addressed.
The study was divided into two parts:
Media Study, Part 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
- Cell growth assessed throughout the time of the experiment
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
- Independent Laboratory – UK Stem Cell Bank, NIBSC. UK – Glyn Stacey
Results of Part 1
The study found that the non-commercial media formulations performed poorly when compared to the commercial products. The likely reason is that commercial products. The likely reason is that commercial formulations are extensively tested for robustness in culturing human ES cells. Non-commercial formulations tend to be simpler and therefore are less tolerant of sub-optimal starting cultures and passaging with very small clumps.
Download a copy of the protocols used in the 1st phase of ISCI-2 [pdf 562kb]
Media Study, Part 2
In addition to media formulation testing described above a second set experiments, coordinated by Peter Zandstra, University of Toronto, has examined what components common to the reported growth media are in fact critical hES cell self-renewal. A manuscript describing these results will be submitted for publication shortly.
Genetic Stability Study
As part of the ongoing efforts started in ISCI-1 to characterize further human embryonic stem cell isolates a prospective study was undertaken to catalogue the range of karyotypic changes that are seen in hES isolates worldwide. The study provides a more definitive picture of common karyotypic changes and minimal amplicons. Chromosome spreads for karyotype analysis and genomic DNA samples, for methylation and SNP analyses were obtained from pairs of hES cell cultures separated by a number of cell divisions.
Samples from 125 independent hES cell lines were provided by 38 laboratories in 19 countries around the world for the study. A small number of hips cell lines were also included.
Results of Part 2
- Human ES cells as a group are ethnically diverse
- Most cell lines remained karyotupically normal but there was an increased frequency of karyotypic abnormalities the longer the cells remained in culture
- Chromosomes 1, 12, 17 and 20 were most likely to be associated with chromosomal gain
- Three regions showed recurrent loss: 10p13-pter, 18q21-qter, 22q13-qter
- A minimal amplicon was identified at 20q11.21 which contains three candidate genes that could promote a selective advantage in hES cells: ID1, BCL2L1 and HM13
- Cell lines carrying the anti-apoptotic Bcl-xl encoded by the BCL2L1 locus, or cell lines overexpressing Bcl-xl possessed a selective growth advantage when compared to their wild-type counterparts