The ISCF brought together an expert working group to lead its international initiative to draw up globally agreed criteria for characterising stem cell lines, focusing on human embryonic stem (hES) lines.
Our working group included experts in stem cell research and banking from Australia, Canada, Israel, Sweden, the USA and the UK, and liaised closely with the Stem Cell Characterisation Unit at the NIH.
Determining the key criteria for characterisation
The characterisation project involved a comparative study of the different hES cell isolates that had been collected worldwide. It was expected that the results would enable the working group to reach a consensus about the key criteria that should be used to identify hES cells, and to establish the degree of heterogeneity that may arise because of different genotypes, different isolation and culture protocols, or because of long term adaptation to culture.
Read the project overview for details of how the project was organised worldwide and how the stem cell lines were characterised. The conclusions are now published in Nature Biotechnology (Adewumi et. al., 2007) and results are available in the ISCI Registry which also includes detailed protocols used in the different parts of the study.
The characterisation project was organised on a ‘hub-and-spoke’ principle. The hub, the UK Stem Cell Bank, collected and prepared antibodies in research-grade facilities for distribution to participating laboratories throughout the world. These laboratories used the antibodies in specified assays of their cell lines under defined conditions. With agreement of the Institutions owning the different hybridomas, this hybridoma collection at the UK Stem Cell Bank provides an archive stock of key reagents for future reference.
The laboratories provided the hub with cell samples (eg, DNA, RNA and protein, as well as xenograft tumour specimens and fixed cells for immunostaining) according to a specified protocol. The hub then sent the samples to central reference laboratories which undertook to assess the expression of selected genes, provide a DNA fingerprint and to assess microbiological status. Several specialist reference laboratories were engaged to carry out specific assays.
For each stem cell line the plan was to:
1. Establish the expression patterns of
- selected surface antigens
- genes marking undifferentiated stem cells and specific lineages of differentiation
2. Compare the changes in marker expression in response to a simple differentiation protocol.
3. Establish the degree of correlation between expression of different potential markers of undifferentiated ES cells.
4. Assess its ‘epigenetic’ status with respect to genes subject to imprinting.
5. Provide a DNA fingerprint of each line.
6. Assess its microbiological status, particularly with respect to endogenous retroviruses.
7. Assess the histology of its xenograft tumours.
8. Collate information regarding origins, karyotype, culture conditions, etc.
Results of ISCI1
Characterized 59 human embryonic stem cell lines from 17 laboratories worldwide
All lines exhibited similar expression paterns, namely expression of:
- SSEA3, SSEA4, TRA-1-60, TRA-1-81, GCTM2, GCT343, CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA,
- Developmentally regulated genes NANOG, POU5F1 (formally known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3.
Lines were not identical:
- Several lineage markers were diferentially expressed.
- Some imprinted genes showed variation in allelic expression.
- Some female lines had reduced expression of XIST in contract to the majority which expressed significant levels.
We did not detect significant contamination of the lines with cytopathic viruses mycoplasma or bacteria.
The results obtained from ISCI1 now form part of the ISCI Stem Cell Registry.
The results are also described in Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nature Biotechnology 25, 803-816 (2007)