Glyn Stacey

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Cryopreservation and freeze-drying Protocols

Contents Preface Contributors Chapter 1 Long-term ex situ conservation of Biological Resources and the role of Biological Resource Centres Glyn N. Stacey and John G. Day Chapter 2 The Process of Freeze-drying Gerald Adams Chapter 3 Principles of Cryopreservation David E. Pegg Chapter 4 Lyophilization of Proteins Paul Matejschuk Chapter 5 Vacuum-drying and Cryopreservation of Prokaryotes Brian J. Tindall Chapter 6 Freeze-drying of Yeast Cultures Chris Bond Chapter 7 Cryopreservation of Yeast Cultures Chris Bond Chapter 8 Freeze-drying Fungi using a Shelf-freeze-drier C. Shu-hui Tan, Cor W. van Ingen and Joost A. Stalpers Chapter 9 Cryopreservation and Freeze-drying of Fungi employing Centrifugal and Shelf Freeze-drying Matthew J. Ryan and David Smith Chapter 10 Cryopreservation of Microalgae and Cyanobacteria John G. Day Chapter 11 Cryopreservation of Plant Cell Suspensions Brian W. W. Grout Chapter 12 Cryopreservation of Shoot-tips and Meristems Erica E. Benson, Keith Harding and Jason W. Johnston Chapter 13 Cryopreservation of Desiccation Tolerant Seeds Hugh W. Pritchard Chapter 14 Cryopreservation of Fish Sperm Eugeny Kopeika, Julia Kopeika and Tiantian Zhang Chapter 15 Cryopreservation of Avian Spermatozoa Graham Wishart Chapter 16 Cryopreservation of Animal and Human Cell Lines Christopher B. Morris Chapter 17 Cryopreservation of Haematopoietic Stem/Progenitor Cells for Therapeutic Use Suzanne M. Watt, Eric Austin and Sue Armitage Chapter 18 Cryopreservation of Human Embryonic Stem Cell Lines Charles J. Hunt and Paula M. Timmons Chapter 19 Cryopreservation of Primary Animal Cell Cultures Glyn N. Stacey and Stuart Dowall Chapter 20 Cryopreservation of Red Blood Cells and Platelets Andreas Sputtek Chapter 21 Cryopreservation of Mammalian Semen Mark R. Curry Chapter 22 Cryopreservation of Mammalian Oocytes Sharon J. Paynter and Barry J. Fuller Chapter 23 Cryopreservation of Mammalian Embryos Barry J. Fuller and Sharon J. Paynter Glossary

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.

Detection of Mycoplasma in cell cultures

Mycoplasma is a prokaryotic organism that is a frequent and occult contaminant of cell cultures. This organism can modify many aspects of cell physiology, rendering experiments that are conducted with contaminated cells worthless. Because of their small size, Mycoplasmas can pass through filters used to prevent bacterial and fungal contamination and potentially spread to all the cultures in a laboratory. It is essential that all new cell cultures entering a laboratory and all cell banks are tested for the presence of Mycoplasma. It is recommended that two techniques be used, selected from a PCR-based method, indirect staining and an agar and broth culture. This protocol describes these three tests for detecting Mycoplasma, which take from 1 d to 3–4 weeks, and such tests should be an obligatory component of quality control in every tissue culture laboratory.

Guidelines for the use of cell lines in biomedical research

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

The International Stem Cell Initiative: toward benchmarks for human embryonic stem cell research

An international consortium is comparing the properties of 75 human embryonic stem cell lines.An international consortium is comparing the properties of 75 human embryonic stem cell lines.

Microbiological control in stem cell banks: approaches to standardisation

The transplant of cells of human origin is an increasingly complex sector of medicine which entails great opportunities for the treatment of a range of diseases. Stem cell banks should assure the quality, traceability and safety of cultures for transplantation and must implement an effective programme to prevent contamination of the final product. In donors, the presence of infectious micro-organisms, like human immunodeficiency virus, hepatitis B virus, hepatitis C virus and human T cell lymphotrophic virus, should be evaluated in addition to the possibility of other new infectious agents (e.g. transmissible spongiform encephalopathies and severe acute respiratory syndrome). The introduction of the nucleic acid amplification can avoid the window period of these viral infections. Contamination from the laboratory environment can be achieved by routine screening for bacteria, fungi, yeast and mycoplasma by European pharmacopoeia tests. Fastidious micro-organisms, and an adventitious or endogenous virus, is a well-known fact that will also have to be considered for processes involving in vitro culture of stem cells. It is also a standard part of current good practice in stem cell banks to carry out routine environmental microbiological monitoring of the cleanrooms where the cell cultures and their products are prepared. The risk of viral contamination from products of animal origin, like bovine serum and mouse fibroblasts as a “feeder layer” for the development of embryonic cell lines, should also be considered. Stem cell lines should be tested for prion particles and a virus of animal origin that assure an acceptable quality.

Changing medium and passaging cell lines

Cell lines are widely used in biomedical research. This protocol describes the methods used routinely to change the medium and passage the cells. Medium changes keep the cells healthy by providing fresh nutrients, while cell passage or splitting is required to maintain cells in exponential growth. Despite the simplicity of the methods used, each cell line has idiosyncracies. Whether working with one or several cell lines, there is no substitute for knowledge of their needs, including the range of phenotypes and growth patterns under different physical and nutrient conditions. Given the necessary care and attention, most cell lines are easy to maintain and grow.

The International Stem Cell Banking Initiative (ISCBI): raising standards to bank on

The International Stem Cell Banking Initiative (ISCBI) aims to create a global network of stem cell banks to facilitate best practice in stem cell research and clinical cell delivery, primary objectives of national and local governments worldwide and stem cell organizations such the International Stem Cell Forum and the International Society of Stem Cell Research. This paper is a brief overview of ISCBI, its primary activities, potential network participants, and the challenges for harmonizing stem cell banking on a global level.

A Call for Standardized Naming and Reporting of Human ESC and iPSC Lines

Human embryonic and induced pluripotent stem cell lines are being generated at a rapid pace and now number in the thousands. We propose a standard nomenclature and suggest the use of a centralized database for all cell line names and a minimum set of information for reporting new derivations.