Safety Testing and Characterisation
The Second International Stem Cell Initiative (ISCI)
Project
One of the first international collaborations funded by ISCF and
co-ordinated by Professor Peter Andrews was the ISCI project. The
first phase of this project was intended to characterise the
features of hESC lines through the collaboration of 17 expert
laboratories from around the world using standardised protocols and
reagents. The final data set on 59 hESC lines from 17 laboratories
was published in Nature Biotechnology in July 2007 (see
Publications). The Bank acted as the technical hub for the project.
It provided reference preparations of antibodies and stable
embryonal carcinoma cells for control purposes, as well as carrying
out all DNA and RNA preparations for dispatch to expert
laboratories within the project. This enabled the characterisation
work, performed in different laboratories on different cell lines,
to be standardised. In addition the Bank provided microbiological
analysis of the stem cell lines.
A second phase of the ISCI project was approved in February 2007
by the International Stem Cell Forum (ISCF). The experimental
programme was agreed at a meeting at the Jackson Laboratories
(Maine, USA) in October 2007 and the first challenge to screen ten
different defined hESC growth media is now under way in four expert
centres in the USA, UK, Japan and Sweden. Analysis of the cultured
cells, again standardised with the support of the Bank as in ISCI
1, will include studies of gene expression (using the Q-PCR low
density array system used in phase 1), flow cytometry for key hESC
markers, genetic stability by SNP analysis and centralised
karyological analysis. After the four laboratories report on
results of the initial growth medium screening process it is
planned to circulate successful media to a large number of
laboratories, with reference reagents and standard protocols, to
examine the media performance in a much broader range of cell lines
in different laboratories. The Bank is again playing a central role
in this project through the provision of reference antibodies, and
RNA control material for expression profiling studies. The Bank is
also developing fixed-cell reference materials for flow
cytometry.
Further information is available on the ISCI page of
the ISCF website
Characterisation of HESCs: Validation of Low Density
Arrays
ISCI 1 explored the expression of a set of 90 genes involved in
hESC pluripotency and differentiation. This set of genes was
interrogated by means of a low density array micro-fluidics gene
card developed by Applied Biosystems. The array enabled a relative
quantification of the gene products and it is now commercially
available (Human ES ‘pluripotency' card). The UKSCB, in its QC
process, currently uses a commercially available RT-PCR stem cell
pluripotency kit that looks at 12 genes. However, the Bank feels
that it could obtain a better gene expression signature to gauge
the quality of the lines, if we employed a quantitative approach
using the gene card. Therefore, the Bank is currently validating
the card for use with the hESC lines using the Applied Biosystems
7900HT system recently purchased by NIBSC.
Characterising Genetic Stability: Comparative Genomic
Hybridisation
Traditionally, karyotyping takes the form of G-banding. This
method can be quite inconsistent, relies on interpretation by a
highly skilled member of staff, is labour intensive and limited in
its capacity to detect genetic changes (i.e. it can only detect
deletions of greater than 10 Megabases (Mb)). In recent years, a
number of laboratories have moved towards comparative genomic
hybridisation (CGH) by microarray as a means to look at chromosomal
aberrations. This technique has a number of advantages, depending
on the type of CGH employed: deletions of as little as 35kb can be
detected; the whole population of cells can be analysed and the
results of the karyotype can be readily interpreted with little
training. A disadvantage is that unlike G-banding, balanced
translocations cannot be detected. It is well reported that cells
in extended passage undergo genetic changes; indeed in the case of
hESC there appears to be a consistent set of chromosomes that are
affected. The Bank would like to move to CGH (initially using a
chip that detects deletions of greater than 0.5Mb) as a means to
monitor cell lines as part of the QC process. This will allow the
Bank to obtain a better picture of the population dynamic with
regard to chromosomal stability. CGH will be included in a number
of projects, initially to look at the feasibility of using this
technique and then to monitor the consistency of the results
obtained using the technology.
Safety Testing of HESCs: Testing for Adventitious Agents
As part of the QC process research grade cell lines are tested
for HIV, HTLV I and II, CMV, EBV, HCV and HBV. In addition, they
are tested for mycoplasma, bacteria, yeast and fungi. The advent of
clinical grade lines may require a more extensive testing regime
for adventitious agents. Using current methods, this can be very
costly and time consuming and consequently the Bank has entered
into collaboration with Applied Biosystems and NIBSC Division of
Virology to explore the development of a micro-fluidics card-based,
low density array of genes to detect the presence of a series of
adventitious agents. This methodology, as with the hESC card,
provides a means of relative quantification of the micro-organisms
of interest. At present, the Bank is in the initial stages of
developing this technology. The Bank is currently exploring limits
of detection using reference standards provided by NIBSC and
sourcing positive controls as each target requires separate
validation before the card is produced.
Validation of an Enhanced Method for Mycoplasma Detection
Current mycoplasma testing procedures in the UKSCB involve
screening the stem cell lines, using direct PCR and broth/agar
culture, at various stages in the banking process. This screening
process during the quarantine period ensures that contaminated
cells new to the Bank are not brought into areas where ‘clean'
cells are in use and that ‘clean' cell lines cannot acquire
mycoplasma contamination during their time in culture in the Bank.
The test sample is generally a small volume (less than 1ml) of cell
suspension or culture supernatant, which may only have been in
contact with the stem cells for 2-3 days. Occasionally, cell lines
will show intermittent positive results by PCR: one test giving a
positive result, with subsequent tests a few days later producing a
negative result. This is thought to be due to low level mycoplasma
contamination: requiring detection by a more highly sensitive test.
Inoculation of the test sample to Vero cell cultures for a period
of 3-7 days has been shown to enrich low level contaminating
mycoplasma (some of which may not grow in mycoplasma isolation
media) and the samples incubated on Vero cells can then be screened
using the current test methods. Assay sensitivity is potentially
greatly increased for both the direct PCR & broth/agar culture
methods following this enrichment step. This work will be
progressed in 2008 with the aim of assessing the sensitivity of the
current mycoplasma testing methods (in terms of cfu/ml detected)
and determining the increase in sensitivity gained using this Vero
amplification step.