International Stem Cell Initiative Research Articles

The International Stem Cell Banking Initiative (ISCBI)

The International Stem Cell Banking Initiative(ISCBI) was started in 2007 to bring together the leading stem cell banks distributing human pluripotent stem cell (hPSC) lines for research and development, to discuss best practice across a range of issues from donor consent to delivery of cells for use in research, diagnostics and cell-based medicines. ISCBI holds workshops around the world and on-line and regularly publishes summaries of discussions and consensus amongst experts in stem cell biology, biobanking technology, regulation and policy making. To date, experts from more than 28 countries have contributed to ISCBI activities which are frequently run in collaboration with other stem cell organisations and has co-ordinated closely with the International Stem Cell Initiative and the hPSCreg European Commission funded database of hPSC lines and clincal trials.

Elevated Exogenous Pyruvate Potentiates Mesodermal Differentiation through Metabolic Modulation and AMPK/mTOR Pathway in Human Embryonic Stem Cells.

SummaryPyruvate is a key metabolite in glycolysis and the tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell-fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance toward oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA-cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism but also modulates signaling pathways in hESC differentiation.

Defining Human Pluripotency.

Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.

Characterization of new variant human ES line V[sbnd]H9 hESC (INSTEMe001-a): a tool for human stem cell and cancer research

Human pluripotent stem cells (hPSCs) acquire changes at the genomic level upon proliferation and differentiation (Peterson and Loring, 2014). Studies from International Stem Cell Initiative and independent laboratories identified a copy number variant (CNV) in hES cell lines displaying a normal karyotype, which provided a selective advantage to hES cells in culture. In our laboratory we have identified variant H9-hESC (derived from H9-hESC) with normal karyotype, pluripotency expression, differentiation profile but with altered traits of high cell survival and low E-CADHERIN expression.

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells

SummaryWe show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes’ expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.

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.

Assessing the Safety of Human Pluripotent Stem Cells and Their Derivatives for Clinical Applications.

Pluripotent stem cells may acquire genetic and epigenetic variants during culture following their derivation. At a conference organized by the International Stem Cell Initiative, and held at The Jackson Laboratory, Bar Harbor, Maine, October 2016, participants discussed how the appearance of such variants can be monitored and minimized and, crucially, how their significance for the safety of therapeutic applications of these cells can be assessed. A strong recommendation from the meeting was that an international advisory group should be set up to review the genetic and epigenetic changes observed in human pluripotent stem cell lines and establish a framework for evaluating the risks that they may pose for clinical use.

Honoring the work and life of Leroy C. Stevens. A symposium as part of the International Stem Cell Initiative Workshop.

In 2016, a symposium was convened in Leroy C. Stevens' honor, in association with a meeting of the International Stem Cell Initiative (ISCI). ISCI, funded internationally, is composed of a group of ~100 scientists from many countries, under the leadership of Peter Andrews, who have worked together to characterize a significant number of human pluripotent stem cell lines, to monitor their genetic stability and their differentiation into mature cell types and tissues in vitro and in vivo. Those at the ISCI meeting puzzled through one of the thorniest problems in the therapeutic use of the differentiated derivatives of pluripotent stem cells for human therapy; namely, pluripotent stem cells can differentiate into any cell type in the adult organism, but they also have the capacity for unlimited self-renewal, hence if mutated they may have tumorigenic potential. The meeting considered how these cells might become genetically or epigenetically abnormal and how the safety of these cells for human therapeutic uses could be assessed and assured. The symposium was an opportunity to pay tribute to Leroy Stevens and to the basic science origins of this newest aspect of regenerative medicine. It was a time to reflect on the past and on how it can influence the future of our field.

BCL-XL Mediates the Strong Selective Advantage of a 20q11.21 Amplification Commonly Found in Human Embryonic Stem Cell Cultures

SummaryHuman embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture, raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV, we show that those containing this amplicon have higher population doubling rates, attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs, only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells, whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells, establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas, linking this mutation with malignant transformation.

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells.

Banking on [Pluri] Potential

Wrestling with issues of provenance, pluripotency, and performance, stem cell banks do more than just freeze and ship cell vials.

Out of Flatworld

BioTechniquesVol. 48, No. 4 Cell Culture: IntroductionOpen AccessOut of FlatworldThe EditorsThe Editors*E-mail Address: bioeditor@biotechniques.comBioTechniquesSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000113420AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Cell culture has become an integral part of both basic research and clinical drug development. The ability to isolate cells, maintain them in culture, and study their biology in vitro has already opened windows into the cellular world. But despite the tremendous number of papers published on culturing cells, the impressive array of commercially available media and reagents, and the utility of cell lines for drug discovery, there still exists a need to develop new and better approaches for culturing a myriad of cell types. With this goal in mind, advances are being made in laboratories around the globe. In this focus section, we have put together a series of articles that examine these needs and describe these recent advances in cell culture technology.Although rich in basic information, it has become clear that culturing cells on flat, plastic dishes is not the optimal approach for understanding how cells work in vivo. Cells in their natural environment interact with a number of factors—including other cells and a variety of chemical and mechanical cues—to drive essential biological processes. It is becoming apparent that understanding processes such as metastasis or stem cell differentiation will require the emulation of cells' microenvironments as accurately as possible in a cell culture dish. But this is no simple task. Matrices that support 3-D growth, as well as isolated growth factors and other chemicals, are now available to add to cell cultures, but determining the optimal combination of these factors to meet certain goals— such as driving differentiation or increasing cell motility—is as much an art at the moment as it is science.Matthias Lutolf and Stefan Kobel examine this issue directly in a review article focusing on the use of high-throughput methodologies to better define stem cell niches. The authors describe advances in microfabrication and biomaterials that are creating cellular microenvironments in wells and dishes that enable a deeper understanding of stem cell biology. From co-culturing cells on three-dimensional scaffolds to exploring the effects of force on single stem cells, Lutolf and Kobel provide an elegant and comprehensive over view of new ways to look at stem cell life in vitro.New stem cell culturing tools, loosening restrictions on the use of embryonic stem cells, and the expansion of induced pluripotent cells have all increased accessibility to stem cell research. And, with the help of stem cell banks, scientists who had avoided using these cells previously are now entering the fray. In a special news article, Jeffrey Perkel takes a look at the emergence of these banks around the globe and examines the lengths to which some go to validate their cell lines for researchers. Although there are no set standard tests yet for such validation efforts by banks, researchers from the International Stem Cell Initiative discuss their ongoing attempts to arrive at a consensus on a set of validation tests for human embryonic and adult stem cell lines.This growing interest in using stem cells, coupled with their increasing availability through stem cell banks and the development of new high-throughput culture approaches as described by Lutolf and Kobel, is making access to more advanced cell culture platforms a reality for all researchers. Expanded numbers of these culture systems and approaches will allow investigators to study their cells under more life-like conditions, and thereby extract the critical information that will not only expand our understanding of cell biology and cell life in diverse microenvironments, but eventually lead to novel therapeutic applications.In the end, we are confident that the articles presented in this focus will provide readers with a strong over view of the latest advances in cell culture technologies, both for stem cells and various other cell types, and provide insight on where these advances will take us in the future.FiguresReferencesRelatedDetails Vol. 48, No. 4 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print April 2010 Information© 2010 Author(s)PDF download

Human ES cell lines—introduction

In the 10 years or so since the first human embryonic stem (ES) cells were described by Thomson and his colleagues (1998), the pace of research with these pluripotent human stem cells has greatly accelerated and is now further stimulated by the development of induced pluripotent (iPS) cells (Takahashi and Yamanaka 2006; Yu et al. 2007). By the time the International Stem Cell Initiative (ISCI) was established in 2004, over 100 human ES cell lines had been established in different laboratories around the world (Andrews et al. 2005); 59 lines were included in the characterisation study of ISCI-1 (Adewumi et al. 2007). Now, over 600 lines are entered into the European Human Embryonic Stem Cell Registry (www. hescreg.eu). One problem this poses for researchers is that simple descriptions of the derivation of new ES cell lines that they produce are difficult to publish alone. Yet such basic descriptions are important background for their future use, but may be difficult to incorporate adequately into articles describing their application in specific experiments. Helpfully, however, The Society for In Vitro Biology does provide a forum in the journal “In Vitro Cellular and Developmental Biology—Animal” where researchers can provide descriptions of the derivation of cell lines that they will subsequently use in later work. In this issue of “In Vitro Cellular & Developmental Biology—Animal,” we are grateful to the Society and

A high-resolution molecular-based panel of assays for identification and characterization of human embryonic stem cell lines

Meticulous characterization of human embryonic stem cells (hESC) is critical to their eventual use in cell-based therapies, particularly in view of the diverse methods for derivation and maintenance of these cell lines. However, characterization methods are generally not standardized and many currently used assays are subjective, making dependable and direct comparison of cell lines difficult. In order to address this problem, we selected 10 molecular-based high-resolution assays as components of a panel for characterization of hESC. The selection of the assays was primarily based on their quantitative or objective (rather than subjective) nature. We demonstrate the efficacy of this panel by characterizing 4 hESC lines, derived in two different laboratories using different derivation techniques, as pathogen free, genetically stable, and able to differentiate into derivatives of all three germ layers. Our panel expands and refines a characterization panel previously proposed by the International Stem Cell Initiative and is another step toward standardized hESC characterization and quality control, a crucial element of successful hESC research and clinical translation.

OCT4B1 isoform: the novel OCT4 alternative spliced variant as a putative marker of stemness

A novel OCT4 alternative spliced variant (OCT 4B1) is deduced to be the isoform present in 59 hESC lines characterised by the International Stem Cell Initiative (ISCI) rather than OCT4A as previously assumed. The new variant may be a more reliable marker of stemness than OCT4A and studies are needed to test this.

Governance-by-standards in the field of stem cells: managing uncertainty in the world of “basic innovation”

This paper explores the role of the International Stem Cell Initiative (ISCI) in its attempt to develop biological standards and experimental protocols for the embryonic field as a whole. Drawing on empirical research derived from UK and other labs and fieldwork within ISCI meetings, we describe the way standards have emerged and the difficulties in stabilizing them at the local level. The paper discusses the ways in which this form of governance-by-standards acts to manage uncertainty – to a degree – and suggests that this requirement to manage uncertainty is becoming a key characteristic of what we call “basic innovation”. We conclude by drawing out the implications of this for the management of uncertainty and the increasingly diverse regulatory space that will be needed to oversee the eventual clinical application of hESC.

Standardizing the Unknown: Practicable Pluripotency as Doable Futures

To standardize human embryonic stem cells is an exercise in standardizing different kinds of unknowns. Such standards, currently being developed in the field, can change the understanding of what a stem cell is. In the influential International Stem Cell Initiative (ISCI), scientists in a fiercely competitive field are prepared to exchange research material and data that would normally be highly confidential. ISCI participants understand the particular unknown that they are seeking to standardize as a ‘known unknown’ and hope that their collaborative work will serve to move the field forward and thus enable both competition and comparable data. Such known unknowns are seen to be of vital importance, yet of a different epistemic currency than the types of unknowns that could lead to scientific fame and fortune. Furthermore, while the notion of ‘pluripotency’ is of pivotal importance as a discursive resource when demarcating the abilities of embryonic stem cells from those of adult stem cells, it can also present a practical problem. A more flexible definition allowing for different stem cell ‘niches’ could render the cell lines less pluri but more potent. The reconfiguration of pluripotency may serve to transport human embryonic stem cells into a clinical and ‘doable’ future.

Proteome Biology of Stem Cells: A New Joint HUPO and ISSCR Initiative

The HUPO initiative “Proteome biology of stem cells” has been established as a collaborative platform bringing together stem cell biologists and researchers in proteomics. The aim is to effectuate the implementation of cutting edge proteomics technology in stem cell research to further our understanding of stem cell biology. This has been prompted primarily by the recent major breakthroughs in stem cell biology, the potential that stem cells have for biomedical application, and the awareness that proteomics has a role in accelerating this progress further or to open as yet unexplored areas. The increasing interest in the characterization and function of stem cells is primarily related to the notion that human embryonic stem cells are highly flexible and have the unique property to form all cells in the human body. The control of this property in vitro potentially offers opportunities to develop treatments of diseases, especially in the area of regenerative medicine, or to design strategies for screening of drugs. Apart from these applications in the long term, molecular understanding of mechanisms controlling stem cell maintenance and differentiation would be of high value for obtaining a basic understanding of the distinctive properties of stem cells. Yet basic as well as clinically orientated research into stem cells is confronted with many challenges and open questions. For instance, cell surface proteins and signaling cascades (both for primitive stem cells as well as differentiated subpopulations) are largely unknown as are differentiation-specific proteins that can be used as benchmarks for the intermediate or terminal steps of differentiation of cells. These are all areas where proteomics can contribute significantly, and given the current state of technology in proteomics, which has matured immensely in recent years (1, 2), it was realized that the time is right to join forces and start a collaborative platform between stem cell biologists and proteomics specialists. As such a common podium for discussion is much required because at present stem cell biology and proteomics communities rarely meet. Therefore, a worldwide alliance has been formed, consisting of leading proteomics and stem cell researchers, resulting in the foundation of an initiative supported by HUPO, named Proteome biology of stem cells early 2007, chaired by Albert Heck and co-chaired by the other authors of this paper (3). Importantly the International Society for Stem Cell Research (ISSCR) immediately recognized the significance of the alliance and has been highly supportive in establishing the initiative and providing a platform to convene, now making this a joint HUPO-ISSCR effort. The inaugural meeting of the consortium was at the annual conference of the ISSCR in Cairns, Australia (June 17–20, 2007). At that occasion, it was concluded that more frequent meetings would be necessary to define appropriate collaborative projects that would be biologically relevant, technically achievable, and productive within a reasonable time frame. Therefore, even at this early stage of the initiative, there have been several rounds of discussions at subsequent meetings of HUPO (Seoul, October 6–10, 2007) and of the International Stem Cell Initiative in Bar Harbor (October 19–21, 2007) attended by investigators from both HUPO and ISSCR organizations. So far, a number of areas have been defined on which collaborative projects could focus. These include the identification of new protein cell surface markers that may be used as fingerprints for stem cell lines or as characteristic marks for cells at specific stages of differentiation. In addition, in-depth analysis of stem cell-specific signaling, for instance through comprehensive phosphoproteomics analyses, would allow a more detailed understanding of stem cell-specific behavior. Over the next few months the discussion will focus on narrowing down specific questions, such as the number and identity of cell lines that should be included in such an initiative, the best growth media and culture conditions, the proteomics techniques that should be applied, and which laboratories would be interested in contributing to such studies. It was agreed that the next annual meeting of ISSCR (Philadelphia, June 2008) would be used to materialize these maturing ideas in a draft proposal, whereas more definitive decisions could be made at the annual HUPO meeting in Amsterdam (August 2008; www.hupo2008.com). A From the ‡Department of Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, ¶Stem Cell and Leukemia Proteomics Laboratory, Faculty of Medical and Human Sciences, University of Manchester, Christie Hospital, Manchester M20 9BX, United Kingdom, Institute for Systems Medicine, Gachon University Gil Medical Center, Incheon 406-799, Republic of Korea, **Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, ‡‡Stem Cell Group, Bioprocessing Technology Institute, 20 Biopolis Way, 06-01 Centros, Singapore 138668, Singapore, §§Center for Stem Cell and Regenerative Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, and ¶¶Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands HUPO Views

Status of genomic imprinting in human embryonic stem cells as revealed by a large cohort of independently derived and maintained lines

Investigation of the epigenetic stability of human embryonic stem cells (hESCs) is a crucial step for their use in cell-replacement therapies, as well as for assessing whether hESCs model epigenetic regulation in human pre-implantation cell types. To address these issues, we have examined the expression of imprinted genes in a previous study and more recently in 46 individual hESC lines as part of the International Stem Cell Initiative. Our results show that nearly all hESC lines examined possessed a substantial degree of epigenetic stability, despite differences in genetic background and in their derivation and initial propagation conditions. However, some hESCs did show loss of allele-specific expression, which could have implications for hESC differentiation and epigenetic stability (both in vitro and after clinical transplantation). A benefit of our and other recent studies of genomic imprinting in hESCs was the identification of imprinted genes that provide a useful indication of epigenetic stability. SNRPN, IPW and KCNQ1OT1 were highly stable and thus appeared insensitive to perturbation; in contrast, H19, IGF2 and MEG3 were more variable and thus could potentially provide a sensitive indication of epigenetic status. In this review, we examine the differences between imprinted genes in their susceptibility to perturbation and discuss the potential molecular basis for these differences. This examination provides insight into the regulation of genomic imprinting in hESCs and the corresponding peri-implantation stages of human development.

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.