RESUMO
This report summarizes the recent activity of the International Stem Cell Banking Initiative held at Harvard Stem Cell Institute, Boston, MA, USA, on June 18, 2017. In this meeting, we aimed to find consensus on ongoing issues of quality control (QC), safety, and efficacy of human pluripotent stem cell banks and their derivative cell therapy products for the global harmonization. In particular, assays for the QC testing such as pluripotency assays test and general QC testing criteria were intensively discussed. Moreover, the recent activities of global stem cell banking centers and the regulatory bodies were briefly summarized to provide an overview on global developments and issues. Stem Cells 2019;37:1130-1135.
Assuntos
Células-Tronco Pluripotentes/citologia , Células-Tronco/citologia , Bancos de Tecidos/normas , Boston , Terapia Baseada em Transplante de Células e Tecidos/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Cooperação Internacional , Controle de QualidadeRESUMO
Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.
Assuntos
Cromossomos Humanos Par 1 , Células-Tronco Pluripotentes , Humanos , Cromossomos Humanos Par 1/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Técnicas de Cultura de Células/métodos , Apoptose/genética , Células Alimentadoras/citologia , Linhagem Celular , Células CultivadasRESUMO
National Eye Institute recently issued a new Strategic Plan outlining priority research areas for the next 5 years. Starting cell source for deriving stem cell lines is as an area with gaps and opportunities for making progress in regenerative medicine, a key area of emphasis within the NEI Strategic Plan. There is a critical need to understand how starting cell source affects the cell therapy product and what specific manufacturing capabilities and quality control standards are required for autologous vs allogeneic stem cell sources. With the goal of addressing some of these questions, in discussion with the community-at-large, NEI hosted a Town Hall at the Association for Research in Vision and Ophthalmology annual meeting in May 2022. This session leveraged recent clinical advances in autologous and allogeneic RPE replacement strategies to develop guidance for upcoming cell therapies for photoreceptors, retinal ganglion cells, and other ocular cell types. Our focus on stem cell-based therapies for RPE underscores the relatively advanced stage of RPE cell therapies to patients with several ongoing clinical trials. Thus, this workshop encouraged lessons learned from the RPE field to help accelerate progress in developing stem cell-based therapies in other ocular tissues. This report provides a synthesis of the key points discussed at the Town Hall and highlights needs and opportunities in ocular regenerative medicine.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Doenças Retinianas , Humanos , Doenças Retinianas/terapia , Doenças Retinianas/metabolismo , Transplante de Células-Tronco , Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Pluripotentes Induzidas/metabolismo , Epitélio Pigmentado da Retina/metabolismoRESUMO
The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.
Assuntos
Pesquisa com Células-Tronco , Humanos , Reprodutibilidade dos TestesRESUMO
It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture in vitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior in vitro or in vivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics.
Assuntos
Epigenômica , Células-Tronco Pluripotentes , Humanos , Pesquisa com Células-Tronco , Oncogenes , Epigênese GenéticaRESUMO
Mouse embryonic fibroblasts (MEFs) can be used in co-culture to support generation of induced pluripotent stem cells (iPSCs) and the normal growth and proliferation of human pluripotent stem cells (hPSCs). Here, we describe the necessary steps to derive, expand, harvest, inactivate, plate, and use MEFs as feeders for iPSC generation and maintenance.
Assuntos
Técnicas de Cultura de Células/métodos , Embrião de Mamíferos/citologia , Células Alimentadoras/efeitos da radiação , Fibroblastos/efeitos da radiação , Animais , Células Cultivadas , Técnicas de Cocultura/métodos , Criopreservação , Células Alimentadoras/citologia , Células Alimentadoras/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , CamundongosRESUMO
We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.
Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Meios de Cultura/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , HumanosRESUMO
Seven human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts from three neonatal individuals using non-integrative reprogramming. Most control iPSCs are derived from adults, so these iPSCs meet the need for control iPSCs from young individuals. Donors were from different ethnicities and these lines provide unique genetic profiles. All iPSCs have normal karyotypes, express stem cell markers, and exhibit pluripotency, as assessed by capacity to differentiate into three germ layers. These lines are valuable to study human development, as age-matched controls for disorder-specific iPSCs, and as platforms for gene editing to control for age and ethnicity.
Assuntos
Técnicas de Cultura de Células/métodos , Etnicidade , Animais , Linhagem Celular , Feminino , Humanos , Recém-Nascido , Masculino , CamundongosRESUMO
November 2018 marks the 20th anniversary of the seminal human embryonic stem cell (hESC) publication, which reported the initial hESC derivations and launched the field of human pluripotent stem cell research. To commemorate this significant milestone, we reflect on the scientific, economic, and clinically relevant impact of this groundbreaking achievement.
Assuntos
Células-Tronco Pluripotentes/citologia , Pesquisa com Células-Tronco/história , Linhagem Celular , História do Século XX , História do Século XXI , HumanosRESUMO
Use of clinical-grade human induced pluripotent stem cell (iPSC) lines as a starting material for the generation of cellular therapeutics requires demonstration of comparability of lines derived from different individuals and in different facilities. This requires agreement on the critical quality attributes of such lines and the assays that should be used. Working from established recommendations and guidance from the International Stem Cell Banking Initiative for human embryonic stem cell banking, and concentrating on those issues more relevant to iPSCs, a series of consensus workshops has made initial recommendations on the minimum dataset required to consider an iPSC line of clinical grade, which are outlined in this report. Continued evolution of this field will likely lead to revision of these guidelines on a regular basis.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/normas , Células-Tronco Pluripotentes Induzidas/citologia , Guias de Prática Clínica como Assunto , Controle de Qualidade , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/microbiologiaRESUMO
Unambiguous cell line authentication is essential to avoid loss of association between data and cells. The risk for loss of references increases with the rapidity that new human pluripotent stem cell (hPSC) lines are generated, exchanged, and implemented. Ideally, a single name should be used as a generally applied reference for each cell line to access and unify cell-related information across publications, cell banks, cell registries, and databases and to ensure scientific reproducibility. We discuss the needs and requirements for such a unique identifier and implement a standard nomenclature for hPSCs, which can be automatically generated and registered by the human pluripotent stem cell registry (hPSCreg). To avoid ambiguities in PSC-line referencing, we strongly urge publishers to demand registration and use of the standard name when publishing research based on hPSC lines.
Assuntos
Bancos de Espécimes Biológicos , Bases de Dados Factuais , Células-Tronco Pluripotentes , Sistema de Registros , Terminologia como Assunto , HumanosRESUMO
The ability of human pluripotent stem cells (hPSCs) to proliferate indefinitely in culture while maintaining their pluripotent properties makes them a powerful tool for use in research, and provides tremendous potential for diagnostic testing, and therapeutic application. Success in these areas, however, is dependent on the ability to effectively expand them in long-term culture while preserving their distinct nature. Contained in this chapter are detailed protocols for the feeder-independent culture and expansion of hPSCs using mTeSR1 medium and Matrigel matrix, and guidelines for the successful transfer of those cells to alternative platforms. These protocols have been used widely by laboratories around the world to successfully expand hPSCs for long-term culture while maintaining their undifferentiated, pluripotent state.
Assuntos
Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Colágeno/metabolismo , Meios de Cultura/metabolismo , Combinação de Medicamentos , Células Alimentadoras/citologia , Células Alimentadoras/metabolismo , Humanos , Laminina/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteoglicanas/metabolismoRESUMO
Cryobanking human pluripotent stem cells (hPSCs), be they human embryonic (hESCs) or induced pluripotent stem cells (iPSCs), is essential for their use in research and cell-based therapeutics. Working and master cell banks can be generated with a desired level of quality assurance applied during cell freezing and storage. Conventional vitrification has evolved to more advanced control rate freezing, culminating in a myriad of published protocols with variable proficiencies and clinical efficacies. Notwithstanding, standardized and reliable protocols are necessary for basic science through to applied research and clinical product development. This chapter details several methods for hPSC cryopreservation, suitable for routine application, high-quality research, and adaptable for clinical compliance.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Criopreservação/métodos , Crioprotetores/farmacologia , Congelamento , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , VitrificaçãoRESUMO
This article summarizes the recent activity of the International Stem Cell Banking Initiative (ISCBI) held at the California Institute for Regenerative Medicine (CIRM) in California (June 26, 2016) and the Korean National Institutes for Health in Korea (October 19-20, 2016). Through the workshops, ISCBI is endeavoring to support a new paradigm for human medicine using pluripotent stem cells (hPSC) for cell therapies. Priority considerations for ISCBI include ensuring the safety and efficacy of a final cell therapy product and quality assured source materials, such as stem cells and primary donor cells. To these ends, ISCBI aims to promote global harmonization on quality and safety control of stem cells for research and the development of starting materials for cell therapies, with regular workshops involving hPSC banking centers, biologists, and regulatory bodies. Here, we provide a brief overview of two such recent activities, with summaries of key issues raised. Stem Cells Translational Medicine 2017;6:1956-1962.
Assuntos
Bancos de Espécimes Biológicos/normas , Células-Tronco Embrionárias Humanas/citologia , Pesquisa com Células-Tronco , Bancos de Espécimes Biológicos/organização & administração , Congressos como Assunto , Humanos , Cooperação InternacionalRESUMO
Data sharing is an essential element of research; however, recent scientific and social developments have challenged conventional methods for protecting privacy. Here we provide guidance for determining data sharing thresholds for human pluripotent stem cell research aimed at a wide range of stakeholders, including research consortia, biorepositories, policy-makers, and funders.
Assuntos
Células-Tronco Pluripotentes/citologia , Privacidade , Pesquisa com Células-Tronco/ética , Humanos , Pesquisa com Células-Tronco/legislação & jurisprudênciaRESUMO
The generation of human embryonic stem cell banking networks has ensured that well-characterized and quality controlled stem cell lines are broadly accessible to researchers worldwide. Here, we provide recommendations for engaging these established networks in efforts to build similar resources for the distribution and collection of induced pluripotent stem cells.
Assuntos
Bancos de Espécimes Biológicos , Técnicas de Cultura de Células , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Linhagem Celular , HumanosRESUMO
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.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Terminologia como Assunto , Linhagem Celular , Humanos , Padrões de ReferênciaRESUMO
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.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Animais , Adesão Celular , Contagem de Células , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Citometria de Fluxo , Humanos , CamundongosRESUMO
In the 10 years that the technology to produce human embryonic stem cell lines has been available, hundreds of lines have been derived in numerous global locations. These cell lines are being used by researchers across diverse scientific fields to investigate the basic biology, clinical potential, and pharmaceutical applications of these cells and their progeny. In this fast-moving and rapidly growing field, how can we ensure that data generated by different laboratories using the same cell lines are comparable, reproducible, and consistent? One suggestion would be to ensure the quality of the "seed stock" material received and used by researchers. Because a number of laboratories worldwide provide stem cell lines to the scientific community, it seems logical to explore the harmonization of practices between distributors to establish cohesive standards and aid the global movement of stem cell lines to the research community. In the future, when these cells arrive in the clinic for therapeutic use, this consensus of "best practice" should ensure the consistency and facilitate the dissemination of these valuable materials.