Lyn Healy

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.

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.

Reconsidering pluripotency tests: Do we still need teratoma assays?

The induction of teratoma in mice by the transplantation of stem cells into extra-uterine sites has been used as a read-out for cellular pluripotency since the initial description of this phenomenon in 1954. Since then, the teratoma assay has remained the assay of choice to demonstrate pluripotency, gaining prominence during the recent hype surrounding human stem cell research. However, the scientific significance of the teratoma assay has been debated due to the fact that transplanted cells are exposed to a non-physiological environment. Since many mice are used for a result that is heavily questioned, it is time to reconsider the teratoma assay from an ethical point of view. Candidate alternatives to the teratoma assay comprise the directed differentiation of pluripotent stem cells into organotypic cells, differentiation of cells in embryoid bodies, the analysis of pluripotency-associated biomarkers with high correlation to the teratoma forming potential of stem cells, predictive epigenetic footprints, or a combination of these technologies. Each of these assays is capable of addressing one or more aspects of pluripotency, however it is essential that these assays are validated to provide an accepted robust, reproducible alternative. In particular, the rapidly expanding number of human induced pluripotent stem cell lines, requires the development of simple, affordable standardized in vitro and in silico assays to reduce the number of animal experiments performed.

Points to consider in the development of seed stocks of pluripotent stem cells for clinical applications: International Stem Cell Banking Initiative (ISCBI)

In 2009 the International Stem Cell Banking Initiative (ISCBI) contributors and the Ethics Working Party of the International Stem Cell Forum published a consensus on principles of best practice for the procurement, cell banking, testing and distribution of human embryonic stem cell (hESC) lines for research purposes [1], which was broadly also applicable to human induced pluripotent stem cell (hiPSC) lines. Here, we revisit this guidance to consider what the requirements would be for delivery of the early seed stocks of stem cell lines intended for clinical applications. The term ‘seed stock’ is used here to describe those cryopreserved stocks of cells established early in the passage history of a pluripotent stem cell line in the lab that derived the line or a stem cell bank, hereafter called the ‘repository’.

The ToxBank Data Warehouse: Supporting the Replacement of In Vivo Repeated Dose Systemic Toxicity Testing.

The aim of the SEURAT‐1 (Safety Evaluation Ultimately Replacing Animal Testing‐1) research cluster, comprised of seven EU FP7 Health projects co‐financed by Cosmetics Europe, is to generate a proof‐of‐concept to show how the latest technologies, systems toxicology and toxicogenomics can be combined to deliver a test replacement for repeated dose systemic toxicity testing on animals. The SEURAT‐1 strategy is to adopt a mode‐of‐action framework to describe repeated dose toxicity, combining in vitro and in silico methods to derive predictions of in vivo toxicity responses. ToxBank is the cross‐cluster infrastructure project whose activities include the development of a data warehouse to provide a web‐accessible shared repository of research data and protocols, a physical compounds repository, reference or “gold compounds” for use across the cluster (available via wiki.toxbank.net), and a reference resource for biomaterials. Core technologies used in the data warehouse include the ISA‐Tab universal data exchange format, REpresentational State Transfer (REST) web services, the W3C Resource Description Framework (RDF) and the OpenTox standards. We describe the design of the data warehouse based on cluster requirements, the implementation based on open standards, and finally the underlying concepts and initial results of a data analysis utilizing public data related to the gold compounds.

Production and validation of a good manufacturing practice grade human fibroblast line for supporting human embryonic stem cell derivation and culture

IntroductionThe development of reproducible methods for deriving human embryonic stem cell (hESC) lines in compliance with good manufacturing practice (GMP) is essential for the development of hESC-based therapies. Although significant progress has been made toward the development of chemically defined conditions for the maintenance and differentiation of hESCs, efficient derivation of new hESCs requires the use of fibroblast feeder cells. However, GMP-grade feeder cell lines validated for hESC derivation are not readily available.MethodsWe derived a fibroblast cell line (NclFed1A) from human foreskin in compliance with GMP standards. Consent was obtained to use the cells for the production of hESCs and to generate induced pluripotent stem cells (iPSCs). We compared the line with a variety of other cell lines for its ability to support derivation and self-renewal of hESCs.ResultsNclFed1A supports efficient rates (33%) of hESC colony formation after explantation of the inner cell mass (ICM) of human blastocysts. This compared favorably with two mouse embryonic fibroblast (MEF) cell lines. NclFed1A also compared favorably with commercially available foreskin fibroblasts and MEFs in promoting proliferation and pluripotency of a number of existing and widely used hESCs. The ability of NclFed1A to maintain self-renewal remained undiminished for up to 28 population doublings from the master cell bank.ConclusionsThe human fibroblast line Ncl1Fed1A, produced in compliance with GMP standards and qualified for derivation and maintenance of hESCs, is a useful resource for the advancement of progress toward hESC-based therapies in regenerative medicine.

Stem Cell Banks: Preserving Cell Lines, Maintaining Genetic Integrity, and Advancing Research

The ability to cryopreserve and successfully recover cell lines has been critical to the conservation of all cell lines, especially the preservation of pristine early-stage cultures and the preparation of well-characterized cell banks. Indeed, the systematic storage and establishment of cryopreserved banks of cells for the stem cell research community is fundamental to the promotion of standardisation in stem cell research and their use in clinical applications. In spite of the significant potential for the use of stem cells in research and therapy, they are challenging to maintain and have been shown to be unstable after prolonged culture that often results in permanent alterations in their genetic make-up, which ultimately alters the phenotype of the culture. This chapter will review the principles of cell bank production, techniques for the scale-up of human pluripotent stem cells, quality control, and characterisation methods for banked cell lines.

Global Solutions to the Challenges of Setting up and Managing a Stem Cell Laboratory

Keywords Stem cell laboratory establishment.Laboratorydesign.Laboratory operation.Good cell culture practice.Laboratory management.Standardization.Quality controlBackground and OriginIn recent years, stem cell research has made major contribu-tions to our understanding of biology. The ability toreprogramme somatic cells to stem cells of desired potencyhas made this exciting area of research accessible to almostevery laboratory, in spite of varying ethical, political andeconomic scenarios. The promise of stem cell research inmaking regenerative medicine accessible has further attractedclinicians, materials scientists, chemists, physicists and othernon-specialists to this field of research. While this situation isdesirable, the novice usually faces the daunting task of settingupandmaintainingastemcelllaboratory,oftenwithoutaccessto local expertise.Researchers allocate a significantamount ofresource to keep their approach technologically advanced.Deliveryofrobustandreliabledata(i.e.achievingreliableandreproducible stem cell cultures for experimentation) is oftenneglected.Thiscanresultindisruptionanddelayinlaboratorywork and at worst, wasted research resource and evenretractionofpublications.Thisdocumentlaysoutfundamentalissues to be addressed in the establishment of a stem cellculture laboratory. The aim is to provide guidance on ways toovercome many challenges to smooth operation, encounteredin varying climates and environments. Parts of this overvieware modeled on the Guidance on Good Cell Culture Practice[3] and should be considered as an ‘aide memoire’ tocomplement existing guidelines. This guidance originatedfrom experience gained by the authors in the establishment ofmultiple cell culture laboratories and training students indifferent countries with widely different environmentalconditions in Northern Europe and in India.ScopeThis document addresses the full range of issues that new aswell as established stem cell researchers charged with settingup a stem cell laboratory may face. It proposes solutions todealwithpotentialproblemsaheadoftime.Theaimistohelpincrease reproducibility of procedures, reduceuncertainties insupply, and help academics meet international scientific andethical requirements.IntroductionGood scientific practice and maintenance of high standardsof mammalian cell culture is important for any researchbased on the use of stem cell lines. Contributions to stemcell research are now global and include researchers andcountries that are relatively new to the field. One importantgoal is to establish consistent standards of scientific andtechnical competence in stem cell culture that will promotegood science and efficient use of research resources. Thispaper identifies generic guidance for establishment and

Beware imposters: MA-1, a novel MALT lymphoma cell line, is misidentified and corresponds to Pfeiffer, a diffuse large B-cell lymphoma cell line

We noted with some anticipation a recent publication in this journal, describing the establishment of a novel mucosa-associated lymphoid tissue (MALT) lymphoma cell line, MA-1 (Kuo et al., 2011). To our knowledge, this would be the first cell line to be successfully established from gastric MALT lymphoma, an unusual extranodal form of B-cell lymphoma. MALT lymphoma has been extensively characterized over the last several decades, but further discoveries are hampered by lack of access to appropriate in vitro models for the disorder (Du, 2007). Our anticipation was tempered, however, by the knowledge that novel cell lines often do not match up to expectations because of crosscontamination and misidentification. Cultures being handled for prolonged periods—for example, during cell line establishment—can easily be cross-contaminated if cells from another culture are introduced by accident (Capes-Davis et al., 2013). The original culture is almost always overgrown by the imposter cell line, often without the scientist at the microscope being aware of the change. The end result is a misidentified cell line, and a series of publications that relate to the imposter rather than the original tissue, cell type or disease state. Cell line cross-contamination and misidentification are not new. Publications dating from the 1960s to the present day (Masters et al., 2012) have urged the scientific community to thoroughly characterize novel cell lines, and authenticate cell line stocks used in experimental work. Recently, however, a sustained effort has been made to standardize the methods used to authenticate human cell lines. Short tandem repeat (STR) profiling is now accepted as an effective way to authenticate human cell lines. A Standard has been published by the American National Standards Institute (ANSI), giving protocols and guidelines for STR profiling as applied to cell lines (ANSI/ATCC ASN-0002–2011, 2012). The International Cell Line Authentication Committee (ICLAC) was established following publication of the Standard to increase awareness and provide resources to help address these ongoing problems (Masters et al., 2012). The MA-1 cell line was analyzed using STR profiling, and its STR profile was published as part of its characterization (Kuo et al., 2011). However, for a cell line to be authenticated, there is a requirement to compare that profile to another sample—preferably from the same donor. Online databases have been developed to allow comparison to other commonly used cell lines, with results made available for this purpose by cell line repositories worldwide (Dirks et al., 2010). An STR profile for the donor of MA-1 was not made available (Kuo et al., 2011), so we compared the cell line result to several STR profile databases (Dirks et al., 2010). A match was found to the Pfeiffer cell line, held by the American Type Culture Collection (ATCC) and established in 1992 from a patient with nonHodgkin lymphoma (Gabay et al., 1999). Comparison of the two STR profiles shows 100% match across eight core STR loci and the gender marker amelogenin (Table 1). These loci have been shown to unequivocally authenticate 98% of cell line samples when assessed using a dataset of 2,279 samples from four cell banks (Capes-Davis et al., 2013). The MA-1 cell line was subsequently obtained from the authors and retested at the Leibniz Institute German Collection of Microorganisms and Cell Cultures (DSMZ) with identical results. Unless further stock can be found that corresponds to the original donor, we must conclude that the MA-1 cell line is misidentified and cannot be used as a model for MALT lymphoma. Laboratories and cell line repositories worldwide are working to characterize existing cell lines, focusing on authentication and diseasespecific markers and mutations (Ottaviano et al., 2010). This task is made more difficult by lack of

Assessment of Established Techniques to Determine Developmental and Malignant Potential of Human Pluripotent Stem Cells

The International Stem Cell Initiative compared several commonly used approaches to assess human pluripotent stem cells (PSC). PluriTest predicts pluripotency through bioinformatic analysis of the transcriptomes of undifferentiated cells, whereas, embryoid body (EB) formation in vitro and teratoma formation in vivo provide direct tests of differentiation. Here we report that EB assays, analyzed after differentiation under neutral conditions and under conditions promoting differentiation to ectoderm, mesoderm, or endoderm lineages, are sufficient to assess the differentiation potential of PSCs. However, teratoma analysis by histologic examination and by TeratoScore, which estimates differential gene expression in each tumor, not only measures differentiation but also allows insight into a PSC’s malignant potential. Each of the assays can be used to predict pluripotent differentiation potential but, at this stage of assay development, only the teratoma assay provides an assessment of pluripotency and malignant potential, which are both relevant to the pre-clinical safety assessment of PSCs.The International Stem Cell Initiative tests methods in a multisite study to detect pluripotency and teratoma formation (PluriTest, Embryoid Body and Teratoma methods) in human pluripotent stem cells. Here, the authors provide guidelines for their application: only the teratoma assay offers evidence of malignant potential.The International Stem Cell Initiative compared several commonly used approaches to assess human pluripotent stem cells (PSC). PluriTest predicts pluripotency through bioinformatic analysis of the transcriptomes of undifferentiated cells, whereas, embryoid body (EB) formation in vitro and teratoma formation in vivo provide direct tests of differentiation. Here we report that EB assays, analyzed after differentiation under neutral conditions and under conditions promoting differentiation to ectoderm, mesoderm, or endoderm lineages, are sufficient to assess the differentiation potential of PSCs. However, teratoma analysis by histologic examination and by TeratoScore, which estimates differential gene expression in each tumor, not only measures differentiation but also allows insight into a PSC’s malignant potential. Each of the assays can be used to predict pluripotent differentiation potential but, at this stage of assay development, only the teratoma assay provides an assessment of pluripotency and malignant potential, which are both relevant to the pre-clinical safety assessment of PSCs. The International Stem Cell Initiative tests methods in a multisite study to detect pluripotency and teratoma formation (PluriTest, Embryoid Body and Teratoma methods) in human pluripotent stem cells. Here, the authors provide guidelines for their application: only the teratoma assay offers evidence of malignant potential.