ISCI-1

The ISCF brought together an expert working group to lead its international initiative to draw up globally agreed criteria for characterising stem cell lines. The project was planned to include characterising stem cell lines not currently listed in the US National Institutes of Health (NIH) stem cell registry, and focused on embryonic stem (ES) cell 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 human ES 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 human ES 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 Nature Biotechnology 2005 Commentary on the ISCI project

(LINK to Nature Biotechnology 2005 Commetary)
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 (atricle available to read) and results are available in the ISCI Registry which also includes detailed protocols used in the different parts of the study (LINK).

Project Overview

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 will use 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

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.

Basic experimental design

Key project stages

Diagram of Hub and Spoke Activities

Hub and spoke diagram

Diagram of characterisation hub and spoke set up

Participating Laboratories

The participating laboratories fell into two groups: those who had derived their own lines, referred to as “primary laboratories”, and those who had obtained their lines from elsewhere, referred to as “secondary” laboratories. You can browse the stem cell lines by the laboratories who worked on them.

The ISCF working group made the final decision about which laboratories and cell lines were included in the project, to ensure that the scale of the study was kept within manageable bounds and to balance the number of cell lines from different sources.

Primary Laboratories

These laboratories carried out all designated experiments with live cells.
They were asked to:

Secondary Laboratories

By involving secondary laboratories we planned to gain some insights into how robust and consistent results were when the same cell lines were cultured by different laboratories.

Pro forma agreement for participants

All data generated by the project is now publicly available on this website. A pro forma agreement was developed for participating laboratories to sign covering: owner permission; ethical standards of cell line derivation; legal liability; and acknowledgement that data generated by the project would be made publicly available.

Central Facilities

UK Stem Cell Bank

The UK Stem Cell Bank and the UK National Institute for Biological Standards (NISB) provided the following services:

Acting as a central receiving ‘hub’

Received materials from participating laboratories and distributed to analytical labs. All material was routed via NIBSC for encoding, which importantly enabled interpretation to be performed blind, with the identity of individual cell lines remaining confidential.

Microbiological analyses

Undertook assays for mycoplasma, known pathogens and endogenous retroviruses.

Antibody (Antigen) Marks: Source/Owner Reference (details here)
TRA-1-60   hES Cells  Wistar  Andrews et al. 1984b
TRA-1-81   hES Cells Wistar  Andrews et al. 1984b
GCTM2     hES Cells ESI/ Pera  Pera et al. 1988
MC631 (SSEA3)  hES Cells Wistar  Shevinsky et al. 1982
MC813-70 (SSEA4)   hES Cells Wistar  Kannagi et al. 1983
TRA-2-49 (L-ALP)   hES Cells Wistar 

Andrews et al. 1984a
TRA-2-54 (L-ALP)   hES Cells Wistar  Andrews et al. 1984b
TG30 (CD9)  hES Cells Pera 
Thy1*   hES Cells Fabre/ICH London McKenzie and Fabre 1981
MC480 (SSEA1)     Diff Cells  ATCC  Solter and Knowles 1978
VINIS56 (GD3)    Diff Cells  Wistar  Andrews and Oliver 1990
VIN2PB22 (GD2)  Diff Cells  Wistar Andrews et al. 1990
B159 (NCAM)   Diff Cells  Wistar 
W6/32 (HLA)   hES Cells ATCC  Barnstable et al. 1978

* For the ISCI1 project, anti-Thy1 from this hybridoma was provided by Chemicon

 

Monoclonal antibodies

Reference cell line

Maintainance of a reference stock of 2102Ep human EC cells as a standard for key surface antigens of undifferentiated human ES/EC cells

Other Central Activities

Gene expression patterns

A central analytical laboratory  carried out gene expression testing and DNA fingerprinting.

  1. Markers of undifferentiated ES cells, eg: Oct4, Nanog, Sox2, TDGF (CRIPTO), FoxD3
  2. Markers of differentiated lineages

A selected panel of 93 genes was analysed by the ABI Microfluidics Real Time RT.PCR system.

In situ immunostaining- M. Pera, Monash University

The spatial distribution of cell types in stock cultures was assessed.

Histopathology - I Damjanov, University of Kansas

Xenograft tumour histology of submitted slides was assessed.

Epigenetic studies  - R. Pedersen, P Rugg-Gunn, University of Cambridge

The expression patterns of several genes that should be expressed monoallelically in ES cells was tested.

Statistical analyses- G. Churchill, B. King, J. Szatkiewicz, W. Zhang, The Jackson Laboratory

The data was analysed to address the questions of whether subsets of hES cells can be identified and which markers provide the best correlates of an undifferentiated hES phenotype.

Results of ISCI1 

The results obtained from ISCI1 now form part of the ISCI Stem Cell Registry.

 

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