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• 29 September 2021

The next frontier for human embryo research

• Elizabeth Svoboda 0

Elizabeth Svoboda is a science writer in San Jose, California.

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In a laboratory in Israel, an incubator drum spins on a bench. The two glass bottles attached to the drum contain mouse embryos, each the size of a grain of rice, with translucent, pulsing hearts.

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Nature 597 , S15-S17 (2021)

doi: https://doi.org/10.1038/d41586-021-02625-0

This article is part of Nature Outlook: Stem cells , an editorially independent supplement produced with the financial support of third parties. About this content .

Aguilera-Castrejon, A. et al. Nature 593 , 119–124 (2021).

Article   PubMed   Google Scholar  

Liu, X. et al. Nature 591 , 627–632 (2021).

Yu, L. et al. Nature 591 , 620–626 (2021).

Tan, T. et al. Cell 184 , 2020–2032 (2021).

Molè, M. A. et al. Nature Commun. 12 , 3679 (2021).

Shahbazi, M. N. et al. Nature Commun. 11 , 3987 (2020).

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• v.16(6); 2021 Jun 8

Human embryo research, stem cell-derived embryo models and in vitro gametogenesis: Considerations leading to the revised ISSCR guidelines

Amander t. clark.

1 University of California, Los Angeles, CA, USA

Ali Brivanlou

2 The Rockefeller University, New York, NY, USA

Jianping Fu

3 The University of Michigan, An Arbor, MI, USA

Kazuto Kato

4 Osaka University, Yamadaoka, Osaka, Japan

Debra Mathews

5 Johns Hopkins University, Baltimore, MD, USA

Kathy K. Niakan

6 Francis Crick Institute and The Centre for Trophoblast Research, University of Cambridge, Cambridge, UK

Nicolas Rivron

7 Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna BioCenter, Vienna, Austria

Mitinori Saitou

8 Kyoto University, Sakyo-ku, Kyoto, Japan

Azim Surani

9 The Gurdon Institute, Cambridge, UK

Fuchou Tang

10 Beijing Advanced Innovation Center for Genomics, Beijing, China

Janet Rossant

11 Gairdner Foundation, Toronto, Ontario, Canada

The ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process.

In this perspective, Rossant, Clark, and colleagues outline the process and deliberations that led to the updated ISSCR Guidelines for stem cell research involving the 14-Day rule, stem cell-based embryo models, and in vitro gametogenesis.

Framing the issues

The ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. At that time, it was already recognized that the ethical issues related to human embryo research extended well beyond the use of human embryos for generation of embryonic stem cells (ESCs). The 2016 guidelines considered broader issues related to human embryo research, including generation of embryos specifically for research, in vitro culture of human embryos, stem cell-embryo chimeras, and genome editing of human embryos. The 2016 guidelines also proposed that all research related to human embryos be subject to oversight by a special process, named Embryo Research Oversight (EMRO), and provided guidance on proposed categories of research that could be allowed, reviewed, or prohibited under such a process.

Since 2016 there has been rapid progress in several areas of human embryo-related research, including technologies for extended in vitro culture of human embryos up to 14 days, creation of stem cell-based embryo models that reflect different stages of human embryo development, and in vitro gametogenesis (IVG) from stem cells. In the light of the changing science, there was a need to revisit the oversight process and the categories of research to be reviewed. A sub-committee of the Task Force to update the ISSCR Guidelines called Working Group 2, was specifically charged with reviewing this area and proposing appropriate revisions to the guidelines. The working group was chaired by Amander Clark and Janet Rossant, and included scientists with relevant expertise and ethicists involved in stem cell/embryo oversight issues (please refer to the author list). The working group had extensive discussions and debates over a 14-month period. Subgroups within this working group focused on particular areas but in the end the entire working group agreed by consensus on the proposed recommendations. These were then further reviewed by the full Guidelines Task Force, the Board of ISSCR, the ISSCR Ethics Committee, and an invited group of regulatory and ethics experts before being subject to external peer review. The final guidelines were approved by the ISSCR Board in December 2020. Guidelines on related research, including germline genome editing and chimera formation were not the purview of Working Group 2, and the deliberative process from these groups are not included here. A white paper on the issues associated with creating chimeric embryos using human stem cells, or contribution of human cells to the germline of chimeras can be found in Hyun et al. (2020) .

General framework

Our working group’s task was to review the existing guidelines and recommend additions and/or modifications to account for the changing science and societal issues. We accepted the general principles underlying the oversight and review process as outlined in the 2016 guidelines, as well as the general concept of a specialized process for review of human embryo- and stem cell-related research. The name “EMRO” was removed from the updated 2021 guidelines recognizing that the specifics of the oversight process would vary in different jurisdictions. Working Group 2 focused on the proposed review categories and the types of research that should fall under each heading.

The 2016 guidelines had three categories of review; the new guidelines divide two of these categories, to provide clearer delineation of the different levels of review.

• New Category 1A: Exempt from review
• New Category 1B: Reportable to an oversight process but normally exempt from review
• 2016 Category 2: Requires review; category is unchanged although the areas of research under this heading have increased
• New Category 3A: Research activities currently not permitted
• New Category 3B: Prohibited research activities

The rationale for these changes, as well as the areas of research that would fall into the different categories are described in more detail in the following sections. It is important to note up front that the culture of human embryos or organized embryo-like structures beyond 14 days, or formation of the primitive streak, whichever occurs first (herein referred to as the “14-day rule”), has been removed from category 3, prohibited activities. This was the subject of many levels of discussion, debate, and consultation over many months. While recognizing that human embryo culture beyond 14 days is prohibited by law or regulation in many jurisdictions, the committee felt that this is an area where a blanket prohibition could inhibit important research directions. The scientific, ethical, and regulatory background to this recommendation is discussed further in later sections.

The decision to update the categories of scientific and ethical review

The decision of the committee to update the laboratory science covered by the different categories reflects not only the changing landscape of stem cell research but also the challenge in defining the concept of “organismal potential,” which was previously proposed as a parameter in reviewing research activities in category 2 or assigning the research to category 3. Furthermore, the committee considered the concept of “time” as part of the 14-day rule to be of limited value when considering the new stem cell-based embryo models given that fertilization is not the starting point to generate a model of the human embryo.

In the case of human stem cell-based embryo models, rather than “organismal potential” we instead proposed a grading of ethical and scientific oversight based on the degree of integration. This is because some embryo models mimic only specific aspects/tissues of human embryo development (non-integrated models), whereas others are designed to model the integrated development of the entire early human conceptus. The models in the first category do not have any reasonable expectations of specifying additional cell types that would result in formation of an integrated embryo model. In contrast, models in the second category might manifest the ability to undergo further integrated development when cultured for additional time in vitro . Therefore, the more integrated the model, the higher the ethical oversight. The committee updated the glossary to define the concepts of an integrated versus non-integrated model of human embryo development.

Based on these discussions, some examples of research activities that should now be considered under the updated categories of review are as follows. For additional examples, please refer to the updated guidelines (ISSCR.org/guidelines).

• a Research with human pluripotent stem cell lines that is confined to cell culture and/or involve routine research practices, such as assays of in vitro differentiation and teratoma formation.
• a Research that entails the in vitro formation of human stem cell-based embryo models that are not intended to represent the integrated development of the entire embryo.
• b Research on IVG from cells, including genetically modified pluripotent stem cells, which does not involve attempts at fertilization and the generation of embryos.
• a Research involving the in vitro culture of human embryos where embryos are maintained in culture until the formation of the primitive streak or 14 days, whichever occurs first.
• b Generation of stem cell-based embryo models that represent the integrated development of the entire embryo, including its extra-embryonic membranes. These integrated stem cell-based embryo models should be maintained in culture for the minimum time necessary to achieve the scientific objective.
• c Research that generates human gametes from any progenitor cell type in vitro , when this entails performing studies of fertilization that produce human zygotes and embryos.
• a The use of human gametes differentiated from human stem cells for the purposes of fertilization and human reproduction.
• b Research in which human embryos that have undergone modification of their nuclear genome are transferred into or gestated in a human uterus.
• a Transfer of human stem cell-based embryo models to the uterus of either a human or animal host.
• b Research in which animal chimeras incorporating human cells with the potential to form human gametes are bred to each other.
• c Transfer of a human embryo(s), irrespective of its origins, to an animal uterus.

In addition to the expanded interest and activity of research using stem cell-based embryo models, the committee also recognized that significant progress has been made with the differentiation of human stem cells and germ cells toward IVG. In the updated guidelines, the committee proposes that IVG should be subject to category 1A and category 1B. However, the formation of embryos after fertilization (or parthenogenesis) of IVG-derived gametes, should require full review under category 2. The use of human gametes differentiated from stem cells for the purposes of human reproduction currently falls under a prohibited activity, category 3A, as the committee decided that safety issues around this technology remain to be resolved. In the following sections, the committee’s deliberations around human embryo models, working with human embryos in culture and the relevance of the 14-day rule, as well as IVG will be discussed.

Human embryo models

Terminology of embryo models.

Over the last few years, human pluripotent stem cells cultured in vitro have demonstrated a capacity to spontaneously organize into structures resembling aspects of the developing early embryo. Because these human embryo models can be formed in large numbers and modified either genetically or physically with greater versatility as compared with human embryos, they represent powerful in vitro assays to understand human embryogenesis and early pregnancy loss. These embryo models do not arise from fertilization or nuclear transfer, they mimic a short developmental window (typically a few days), and in some cases only mimic specific aspects/tissues of human embryo development. As such, stem cell-based embryo models should not be considered equivalent to human embryos under most legislation ( Table 1 ). Considering the proportionality (balancing the benefits and harms) and subsidiarity (pursuing goals using the morally least problematic means) of human embryo research, the committee recognized that embryo models are an ethical alternative to the use of embryos for in vitro research. The revised guidelines have incorporated these embryo models under existing ethical frameworks so as to ensure that research advances in agreement with ethical and societal goals ( Pereira Daoud et al., 2020 ; Hyun et al., 2020 ; Rivron et al., 2018a ; Sawai et al., 2020 ).

Definitions of embryos and human stem cell-based embryo models, including categories under which each embryo type are reviewed

In addition, to best reflect the state and the envisioned applications of these structures made from stem cells, the use of the umbrella term “embryo model” or “stem cell-based embryo model” is encouraged, while the use of the term “synthetic embryo” or “artificial embryo” or “embryoids” should be avoided. Furthermore, the establishment of a terminology precisely reflecting the degree of integration and the type of model is encouraged (e.g., post-implantation amniotic sac embryoid [PACE] [ Zheng et al., 2019 ], blastoid [ Rivron et al., 2018a ]).

Integrated versus non-integrated embryo models

Here, we propose a classification of human embryo models with the aim of guiding the decisions of the scientific and ethical oversight process. The non-integrated embryo models will be models that mimic only specific aspects/tissues of human embryo development and often do not have any associated extra-embryonic membranes. These non-integrated embryo models are reportable but not normally subject to further review (category 1B). In contrast, the integrated embryo models which contain the relevant embryonic and extra-embryonic cell types and could potentially achieve the complexity where they might realistically manifest the ability to undergo further integrated development if cultured for additional time in vitro should be subjected to a full specialized review (category 2). Given that the stem cell-based embryo models are not considered equivalent to human embryos under most legislation (as described in detail above), the decision was made that the integrated embryo models should not be subject to the restrictions of the 14-day rule. In addition, for both ethical and safety reasons, transferring any human embryo model into the uterus of a living animal or human is prohibited (category 3B).

Examples and potential applications

By recapitulating in vitro early human embryonic events, the use of human embryo models for scientific discovery opens ethical alternatives to addressing important biomedical problems. For example, in the next decade, non-integrated human embryo models are likely to model specific events that occur during the first few months of human embryo development, including gastrulation, body axis formation, and somitogenesis, thus allowing the investigation of numerous aspects of embryogenesis-related pregnancy problems and genetically inherited defects. Furthermore, the non-integrated embryo models are likely to help researchers to gain basic knowledge of the specific molecular and cellular events associated with genome mutations associated with developmental origins of disease. They should also guide drug discovery and biomedical strategies aiming at managing genetic diseases or forming or regenerating complex organs for regenerative medicine. Examples of such models include human pluripotent stem cells grown on micropatterned two-dimensional surfaces with confined geometry ( Warmflash et al., 2014 ), gastruloids ( Moris et al., 2020 ; van den Brink et al., 2014 ), PACE ( Zheng et al., 2019 ), or neuruloids ( Haremaki et al., 2019 ).

Stem cell-derived blastoids that mimic the blastocyst stage of development have been produced in the mouse ( Kime et al., 2019 ; Li et al., 2019 ; Rivron et al., 2018b ; Sozen et al., 2019 ) and very recently in the human ( Liu et al., 2021 ; Yanagida et al., 2021 ; Yu et al., 2021 ). In the next decade, such integrated human embryo models are likely to progress from the blastocyst equivalent stage through the steps of early post-implantation development, including human primitive streak formation, gastrulation, formation of the embryonic germlayers, and specification of primordial germ cells (PGCs) thus allowing the study of numerous processes that require interactions between the embryonic and extra-embryonic tissues . Integrated embryo models are likely to guide drug discovery and biomedical strategies aiming at managing early pregnancy to address global health issues, such as infertility as a consequence of unexplained early pregnancy loss, development of new non-hormonal contraception technologies, or formulation of new culture conditions that could be used to improve in vitro fertilization (IVF) culture media.

Working with human embryos in culture, and the relevance of the 14-day rule today

Deliberation process.

Recent technological advances now allow in vitro culture of human embryos for up to 14 days ( Deglincerti et al., 2016 ; Shahbazi et al., 2016 ). These studies and others that followed have unveiled some molecular and cellular events that occur at post-attachment stages of human embryonic development, the discovery of species-specific attributes of early embryo development and have highlighted the limitation of using model organisms in extrapolating information to human embryogenesis ( Gerri et al., 2020 ). For example, the existence of a species-specific yolk sac trophectoderm tissue in human embryos could not have been extrapolated from model systems, such as the mouse ( Deglincerti et al., 2016 ). Similarly, while studies in non-human primates show close comparators with human development ( Nakamura et al., 2017 ; Sasaki et al., 2016 ), equivalence to humans should not be assumed. It has been reported that non-human primate embryos have been successfully cultured up to 21 days, including through the gastrulation period ( Ma et al., 2019 ; Niu et al., 2019 ), suggesting that it should be technically feasible to successfully culture human embryos beyond 14 days.

The 14-day rule has been a broadly adopted limit on the culture of human embryos ( Matthews and Morali 2020 ). This “rule” is in many cases not legally binding and instead is an intended acknowledgment of, and compromise with, the range of strongly and deeply held beliefs about the moral status of human embryos across some, but not all cultures and religions—an effort to allow some scientifically valuable research to move forward, within societally agreed limits ( Hug, 2006 ; Hyun et al., 2016 ; Warnock, 1984 ; Williams and Johnson, 2020 ). Of note, going beyond the 14-day limit never became an active issue until recently, because human embryos could not be kept alive in culture beyond about a week. While the 14-day rule was somewhat arbitrary, it does define a clear developmental window before the body axis and the nervous system begin to form and after which twinning is no longer possible.

Given the technical advances described above, some jurisdictions have begun to reconsider the 14-day rule, motivating the panel to engage in an extensive deliberation about the potential benefits and risks of extending the 14-day rule. The panel was predominantly in favor of extending the redline, although dissenting opinions were also voiced. All of the group ultimately agreed to remove “culture of human embryos beyond 14 days or primitive streak formation” from the category of prohibited activity under category 3. Since the ISSCR Guidelines are only re-evaluated every 5 years, it was felt that now was the time for the community to engage in meaningful and substantial public communication and deliberations. Given advancements in human embryo culture, and the potential for such research to yield beneficial knowledge that promotes human health and wellbeing, national academies of sciences, academic societies, funders, and regulators should lead public conversations on the scientific significance as well as the societal, moral, and ethical issues of allowing such research. It should not be assumed that the public will necessarily support the extension of the 14-day rule, which was historically an important policy position fostering public trust in research and acknowledging broadly held social values. If such conversations do lead to broad public support for the research within a jurisdiction, and if local policies and regulations permit, embryo culture beyond 14 days and into primitive streak formation and gastrulation could be considered in those jurisdictions for review by the specialized oversight process under category 2. Such a review should carefully consider whether the scientific objective of the research justifies the time in culture beyond 14 days and ensure that only a minimal number of human embryos are used to achieve the research objectives. Established ISSCR Guidelines for research projects aimed at illuminating the events up to 14 days post fertilization or before primitive streak formation will remain the same.

To aid in the ongoing debate, we provide some of the considerations that were aired in the committee deliberations on the 14-day rule. The arguments in favor of maintaining the 14-day rule for the time being are that the second week of embryonic development has only recently become accessible for study, and there is still much to be learned between 7 and 14 days post fertilization. In addition, the scientific community should demonstrate for the public the value of the original compromise—What has been learned about the first 7 days of human development? and What impact has the knowledge made on clinical care? The scientific community needs to take the time to justify for the public revisiting the previously agreed compromise. Furthermore, the methodologies for culturing human embryos up to 14 days have recently been developed and may require further optimization, for example to consistently maintain a yolk sac cavity. Arguments in favor of extending the limit were largely based on the potential scientific and clinical benefits. There is a considerable gap of knowledge between the first 2 weeks of human development and the fourth week of life; a time that involves high rates of early pregnancy loss, thus making this stage very challenging yet extremely important to study. Preclinical assessment of this developmental stage would be particularly informative for future advances in mitochondrial replacement therapy, IVG, or germline genome editing. There is an increasing need to perform comparative studies of human embryos to stem cell-derived embryo models, allowing for the assessment of the fidelity of in vitro stem cell-based embryo model systems. If validated, these embryo model systems can be used in the future instead of human embryos to study the cell and molecular events that occur during and after primitive streak formation. There are also several direct clinical implications to studying human embryos beyond the 14-day rule. Early congenital diseases, and some late-onset diseases ( Gluckman et al., 2008 ), have their roots in early embryogenesis. Examples include autism ( Miller et al., 2005 ), heart malformation ( Anderson et al., 1974 ), and neural tube defects ( Greene and Copp, 2014 ). Advances in our understanding of such diseases would require a knowledge of the cellular and molecular events that occur during the development of the nervous system, the heart, and other organs, which would require extending the limits on in vitro culture close to Carnegie stage 12 (day 26–30). In addition, in vitro culture of human embryos would decrease the burden on the experimental use of animals, especially non-human primates. Individuals who donate human preimplantation embryos to research do so following informed consent with counseling available. Donation is nearly uniformly of material that is surplus to IVF treatment, which would be otherwise destroyed, and is often viewed by the donating individual as positively contributing to future clinical improvements.

In summary, the future of human embryo culture beyond 14 days to study gastrulation and post-gastrulation events, such as primitive streak formation, early germ layer development, formation of PGCs, and early organogenesis remains to be determined and will certainly run into different barriers in different jurisdictions. In several countries there is a legal ban on human embryo culture beyond 14 days and there are regulatory restrictions in many others ( Matthews and Morali, 2020 ).

Human IVG—Where to draw the regulatory line today

Generation of gametes in vitro (IVG) from human cells provides the opportunity to study human germ cell development, including the processes of imprint erasure, imprint resetting, and meiosis. Failure to erase and reset imprints can lead to the birth of children with developmental disabilities. Furthermore, aneuploidies arising through meiotic errors can lead to either pregnancy loss or children born with chromosomal conditions leading to morbidity and mortality. Therefore, understanding the process of human germ cell development, including the mechanisms of imprinting and meiosis, are essential to understanding infertility and diseases that impact human reproduction and child health. In this section, the promise of IVG will be highlighted based on work using the mouse. This will be followed by oversight and review considerations for performing IVG with human cells.

The committee’s deliberations on IVG focused on the use of human ESCs (hESCs) and human induced pluripotent stem cells (iPSCs) given the recent success using mouse cells ( Hayashi et al., 2011 , 2012 ; Ohinata et al., 2009 ; Hikabe et al., 2016 ; Zhou et al., 2016 ). Specifically, IVG with mouse ESCs or iPSCs involves first differentiation into epiblast-like cells followed by a second step of differentiation into primordial germ cell-like cells (PGCLCs). PGCLCs are diploid germ cells with the potential to differentiate into oogonia-like cells ( Hikabe et al., 2016 ) or male germline stem cell-like cells (GSCLCs) ( Ishikura et al., 2016 ) that undergo meiosis to become gametes (oocytes or sperm) when cultured or transplanted into an appropriate niche. Notably IVG to create fertilization-competent oocytes requires a final step of in vitro maturation (IVM) before successful fertilization and the birth of healthy offspring ( Hikabe et al., 2016 ). In addition to starting with ESCs and iPSCs, gametes have also been created in vitro from mouse organ cultures using prenatal ovaries ( Morohaku et al., 2016 ); primordial follicle culture followed by IVM ( Eppig and O'Brien, 1996 ; O'Brien et al., 2003 ); and culture of neonatal testis tissue fragments ( Sato et al., 2011a , 2011b ). Translating these in vitro technologies to human cells for basic science research on gametogenesis will require appropriate oversight and review under existing mandates and/or committees for procuring and working with human tissues and cells. Using the human IVG-derived gametes for fertilization to create human embryos will require specialized scientific and ethics oversight as detailed below.

Starting with human ESCs and iPSCs, the initial steps of IVG to generate PGCLCs have been widely reported ( Chen et al., 2017 ; Irie et al., 2015 ; Sasaki et al., 2015 ). In addition, human PGCLCs have the capacity to differentiate into human oogonia-like cells and oocytes ( Yamashiro et al., 2018 ). However, the creation of ovarian follicles containing oocytes equivalent to those found in the adult human ovary remains to be achieved. Furthermore, the differentiation of GSCLCs or sperm from human cells has not been documented. IVG with immature human follicles isolated from the ovary (also called in vitro growth) before IVM is an active area of research. Safety concerns using IVG of follicles before IVM should also be considered as this is a critical window when imprints are re-established and meiosis resumes ( Telfer, 2019 ). Together, these studies indicate that IVG with human cells is promising technology for restoring fertility. Yet a broad societal discussion is still needed, particularly when beginning with human pluripotent stem cells.

Based on this background, the committee recommended that basic research on human IVG without experiments designed to fertilize the resulting gametes should be permissible as a category 1B research activity. Although expected to be rare, it is theoretically feasible that under some circumstances parthenogenetic embryos may spontaneously develop from gametes produced by IVG. Given this, it is suggested that investigators report the creation of IVG-derived parthenogenetic embryos to a specialized scientific and ethics oversight process. This will enable review of the project and a determination as to whether future research should remain category 1B or comprehensively reviewed under category 2.

For scientists engaging in IVG with 46, XX or 46, XY cells where the research project involves IVM and fertilizing gametes to create human IVG-derived embryos, this research is permissible provided that embryos are maintained in vitro only. Such research must be reviewed under category 2 by a specialized scientific and ethics oversight process. Examples of permissible experiments could include the study of IVG-derived embryos up to 14 days post fertilization or formation of the primitive streak, whichever occurs first, or the derivation of cell lines from IVG-derived embryos. Experiments designed to transfer an IVG-derived human embryo into the uterus of a non-human animal host should not be pursued at all. Such experiments would be considered a category 3B activity because of broad international consensus that such experiments lack a compelling scientific rationale and are widely considered to be unethical. Similarly, research into which animal chimeras incorporating human cells with the potential to form human gametes are bred to each other is also considered a category 3B activity.

Finally, there is no compelling scientific evidence that IVG is currently safe for use in human reproduction, particularly when starting with hESCs, iPSCs, or iPSC derivatives, including PGCLCs, oogonia- or oocyte-like cells, or GSCLCs. This is because of unresolved issues related to epigenetic and genetic abnormalities of the resulting gametes, particularly given that the mouse oocytes and mouse GSCLCs derived from stem cells are reported to be of lower quality than their in vivo counterparts ( Hikabe et al., 2016 ; Ishikura et al., 2016 ). Therefore, it was recommended that IVG for human reproductive purposes be categorized as a currently prohibited research activity until safety and ethical issues are resolved (category 3A). It was recognized that this technology will have the potential for use in human reproduction once safety and efficacy is proven, with the most promising approach likely to be IVG from immature follicles collected and frozen as part of fertility preservation before cancer treatment or sterility-inducing bone marrow transplants ( Medicine, 2019 ). Furthermore, IVG and IVM to create sperm from pre-pubertal tissue may not be far behind.

In summary, the 2021 ISSCR Guidelines were updated to include a new regulatory and ethical framework for the oversight of IVG research and the creation of human embryos after IVG. This new framework recognizes the importance of IVG to generate basic science knowledge on the cell and molecular regulation of human germ cell development and human reproduction. In addition, by creating category 3A, the updated guidelines leave open the possibly that IVG could be used in the future to treat infertility if proven safe and remaining ethical issues are resolved.

While recognizing that science moves faster than any set of guidelines and regulations can possibly respond, we hope that the ISSCR 2021 guidelines (ISSCR.org/guidelines) on human embryo research are flexible and far-sighted enough to provide the international community with some thoughtful guidance in considering and reviewing new and important areas of research.

Conflicts of interest

Amander T. Clark is a board member of the ISSCR and a Scientific Advisory Board member of the Tepper Foundation.

Ali Brivanlou is a co-founder or OvaNova Inc., as well as a co-founder of Rumi Scientific Inc.

Debra Mathews is a member of the Maryland Stem Cell Research Commission and a paid Academic Collaborator of the National Academy of Medicine's Committee on Emerging Science, Technology, and Innovation in Health and Medicine.

Nicolas Rivron is an inventor on two patents describing the blastoid technology (EP2986711 and EP21151455.9). He has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Program ERC-Co grant agreement no. 101002317.

Mitinori Saitou is an inventor on patent applications relating to the induction of germ cells from PSCs filed by Kyoto University.

Janet Rossant is a member of the Board of Directors of Notch Therapeutics; a member of the editorial board of Stem Cell Reports; and a member of the editorial board of Cell Stem Cell.

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Current State of Human Embryonic Stem Cell Research: An Overview of Cell Lines and Their Use in Experimental Work

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Anke Guhr, Andreas Kurtz, Kelley Friedgen, Peter Löser, Current State of Human Embryonic Stem Cell Research: An Overview of Cell Lines and Their Use in Experimental Work, Stem Cells , Volume 24, Issue 10, October 2006, Pages 2187–2191, https://doi.org/10.1634/stemcells.2006-0053

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Research in human embryonic stem cells (hESCs) is a rapidly developing scientific field. In this study we collect and evaluate a thorough body of data on the current number of publicly disclosed hESC lines and the extent and impact of scientific work involving the use of these cells. These data contribute to the substantiation of the discussion on the current status of hESC research, provide a basis for the analysis of the status of such research, and uncover further needs in terms of registration, banking, standardization, and tracing.

Since human embryonic stem cell (hESC) lines were first derived in 1998 [ 1 ], these cells have been in high demand as objects of research. The ability of hESCs to reproduce almost limitlessly and to differentiate into many, if not all, cell types of the human body has generated an enormous amount of scientific interest. These unique capabilities provide a means of exploring many promising lines of research, which are likely to reveal a deeper understanding of human cellular biology and which may lead to potential cures for many diseases [ 2 , 3 ]. On the other hand, considerable controversy has arisen regarding this type of research, because derivation of hESCs requires destruction of early human embryos [ 4 ]. Consequently, national legislation regulating research involving hESCs varies widely across many countries [ 5 ]. The present paper offers information that can provide needed substance to the debate on hESC research by presenting comprehensive data on the number of currently existing hESC lines and on the actual extent of experimental work undertaken with these cells and published as of December 2005 worldwide, based on the exploration of verifiable public sources.

Figure 1A provides an overview of the number of hESC lines currently derived and in existence according to published data available from various sources. In total, information on 414 human ES cell lines was available. According to data published as of January 1, 2006, hESC lines have been established in at least 20 countries. Although the number of existing hESC lines is quite impressive, only limited data on characterization of these cell lines are publicly available. Currently it is not clear whether all lines are indeed pluripotent hESC lines. According to our database searches, derivation and at least partial characterization of only 43.2% (179, of which 171 are in English‐language journals) of these cell lines have been published in peer‐reviewed journals, and the use of an additional 6% ( n = 25) has been published without detailed characterization data in peer‐reviewed journals. Consequently, 49.2% of all cell lines have been published so far in scientific journals ( Fig. 1B ). Publication in a peer‐reviewed journal provides some information about the hESC‐like characteristics, but it does not provide absolute certainty on their quality. Especially in cases where multiple cell lines are described in a single study, characterization data are only shown for selected cell lines. Seventy‐one different hESC lines are listed in the NIH Stem Cell Registry. Of these, only 22 are currently available to researchers. Of the remaining 49 hESC lines, only three have been characterized with respect to their stem cell nature and have been published in a peer‐reviewed journal; two additional cell lines were published but have been withdrawn by the providers. Although the vast majority of hESC lines were derived using classic cultivation in the presence of feeder cells, 32 were derived under conditions free of animal cells and media containing animal‐derived serum. However, it has to be taken into account that this is not equivalent to “animal‐free” or “xerofree” conditions, as stated by some authors, since serum replacement used in some of these derivations may also contain animal‐derived compounds. Twenty‐seven hESC lines harbor defined genetic defects characteristic for distinct inheritable genetic disorders, and eight cell lines have been shown to have an abnormal karyotype. In addition to the 414 cell lines, 14 hESC lines have been clonally derived from existing hESC lines, some of which are listed in the NIH registry. Almost no information is publicly available on 106 of the 144 cell lines provided by the Reproductive Genetics Institute in Chicago and available from Stemride International Ltd. Remarkably, there were four times more new hESC lines published in peer‐reviewed journals in 2005 ( n = 88) than obtainable (available) from the NIH ( n = 22). A detailed list of currently existing and publicly known hESC lines (including their respective references) is provided as supplemental online Data.

Derived hESC lines (as of January 1, 2006). Data were extracted from the NIH registry, from work published in scientific journals listed in the PubMed database, and from information either available online, presented at conferences, or provided in press releases. To date, only a portion of these cell lines have been published in scientific journals. No detailed information on the geographic origin of the 144 cell lines derived at the Reproductive Genetics Institute and distributed by STEMRIDE International Ltd. is publicly available from sources used in this study (marked as unknown). hESC lines derived at ES Cell International were assigned to Australia. Detailed information on the hESC lines is shown in supplemental online table. (A): Total number of cell lines sorted by country of origin. (B): Cell lines described or used in work published in peer‐reviewed journals. Abbreviation: hESC, human embryonic stem cell.

We next wished to determine the number of scientific publications reporting on derivation and characterization of hESC lines or their experimental use. To cover all relevant papers on experimental use of hESCs, we performed searches of the PubMed database with no restriction to a publication category (such as “journal article”) introduced into the search criteria. Although such restrictions do not reliably exclude papers without relevance for our study, this practice often results in the exclusion of relevant publications. Therefore, the broader search criteria reported in the online supporting material were applied. The searches resulted in more than 2,500 hits over the period spanning from January 1, 1998 to December 31, 2005. These hits were manually evaluated to exclude those articles in which hESCs were not used experimentally (e.g., reviews, tutorials, news, comments, work on mouse ES cells, etc.). Practical work using human embryonic carcinoma or embryonic germ cells but not hESCs was omitted, as was work in which hESC‐derived materials (e.g., RNA or cDNA) but not hESCs were used. We found a total of 315 research papers describing derivation and/or experimental use of hESC lines that had been published (including online publication ahead of print) through December 31, 2005. Most of these research papers came from groups in the United States, followed by Israel, the U.K., and South Korea ( Fig. 2 ). Interestingly, the number of scientific reviews and papers on ethical or legal aspects of hESC research by far exceeded the number of original publications describing experimental work conducted with these cells, suggesting that discussion about hESC research has outpaced actual research activities and continues to do so. According to the list of impact factors of 2004 (Institute for Scientific Information, Thomson Scientific, Philadelphia, http://www.thomson.com/scientific/scientific.jsp ), the average impact factor for journals that have been publishing experimental work with hESCs was 6.03, indicating that an outstanding interest exists for this kind of research. Approximately 28% of published research focused on differentiation of hESCs into specialized cell or tissue types, with a noticeable emphasis on neural, heart, and blood cells. A comparable number of papers (approximately 27.5%) dealt with the molecular characterization of hESCs, including signal transduction, gene expression patterns, or early differentiation. Another 33% of papers described derivations of hESC lines or the establishment of improved culture conditions. In Table 1 , the top 20 publications in the field (cited most frequently since the establishment of the first stem cell line in 1998) are listed. Table 2 gives an overview of the leading journals in the field of experimental hESC research with respect to the number of published research papers. The complete list of publications detected by our method is available as online supporting material. It is worth noting that according to information provided in the papers, at least 29% ( n = 92) of all studies and 54% ( n = 68) of the U.S. studies were conducted with at least partial financial support from the NIH.

Overview of published work reporting on experimental use of hESCs. Searches of the PubMed database were performed as described in the supplemental online methods, and results were evaluated manually to exclude false‐positive hits. (A): Number of papers extracted from the PubMed database by the described search string and the number of research papers reporting on derivation and/or experimental use of hESC lines. Data were sorted by publication year. Advance online publications available as of December 31, 2005, have been included. (B): Number of publications describing derivation and/or experimental use of hESC lines sorted by location of corresponding authors. Abbreviation: hESC, human embryonic stem cell.

Most frequently cited papers reporting on derivation of or experimental work with hESCs and published from 1998 to 2004

Top journals with respect to publication of experimental work involving human embryonic stem cell (hESC) lines

We have also determined the frequency at which specific hESC lines have been used in published research. Information on the hESC line used was available from 91.4% of studies. In total, 681 uses of hESC lines have been reported in these studies. Notably, 210 cell lines (50.7%) have never been described or used in published experimental work so far, and 150 cell lines (36.2%) were only described or used in a single scientific report. Of the remaining 54 hESC lines (13.1%), only 15 (3.6%) have been used in more than 10 studies published in the period investigated ( Table 3 ). Among these, the cell line H9 and its clonally derived derivates were used most frequently in work (16.1% of all uses), followed by cell line H1 and its clonal derivate H1.1 (13.6%). Our data show that NIH‐approved cell lines have been used in the majority of studies published through 2005. This might be due to the good knowledge of these cell lines, to the high proportion of research funding by NIH in the field, or to the fact that use of NIH‐approved cell lines has been in accordance with regulation in other countries, such as Germany. A considerable portion of work published in 2005 (161 uses/derivations; 43.5% of all published hESC uses in 2005) reported on derivation of novel hESC lines or use of hESC lines not registered at the NIH (supplemental online Fig. 1 ). However, in most cases, the non‐NIH‐approved cell lines were used only in a single study (97 of 161 uses/derivations; 60.2% of reports on use/derivation of novel cell lines in 2005). This clearly suggests that an increasing number of researchers are establishing and using their own hESC lines. Although there is a need for new and easily accessible hESC lines derived under animal‐free conditions, increased use of a multitude of hESC lines in research might diminish the comparability of results. Availability of few well‐characterized and easily accessible hESC lines derived under animal‐free conditions and provided by specialized institutions, such as stem cell banks, might be an alternative for research.

Most frequently used human embryonic stem cell (hESC) lines

While this paper was under review, Owen‐Smith and McCormick [ 6 ] published a study of experimental work performed by the end of 2004 and using hESCs. As a basis for their study, they used those reports that cited the first derivation of hESCs by Thomson et al. [ 1 ] 1998. Although some of their data is in good agreement with our findings, there are also some notable differences. For example, we found evidence for 91 ES cell lines published by the end of 2004 in peer‐reviewed papers, whereas Owen‐Smith and McCormick [ 6 ] reported 70 hESC lines. This might be due to differences in the initial search method and to the different databases used for the studies. For example, 18.5% (58) of the papers detected by our search method did not cite the work of Thomson et al. [ 1 ]. In addition, whereas Owen‐Smith and McCormick [ 6 ] conclude that U.S. research started to lag behind international hESC research in the last years, our data revealed that 43% ( n = 40) of hESC papers published in 2004 came from U.S. groups. Similarly, in 2005, 38% ( n = 62) of hESC papers were published by researchers located in the United States. These divergent findings are probably due to the fact that international collaborations of U.S. groups have been marked as “collaborative research” by Owen‐Smith and McCormick [ 6 ]. Because there is an ongoing globalization of hESC research, we considered the localization of the corresponding author's laboratory as more appropriate for assigning a study on hESCs to a country.

In summary, we provide data on the current state of experimental research involving human embryonic stem cells. Although the completeness of this review is contingent upon the limitations of the search methodology, incomplete information in some published articles, and the lack of accepted registry and tracing mechanisms for hESC lines, we provide evidence that hESC research is a field that has been developing rapidly, especially within the last 3 years. Although the number of published hESC lines has markedly increased within the last 3 years, most published research has been performed with cell lines derived before the end of 2001. However, the growing number of a variety of well‐characterized new hESC lines partially harboring defined genetic defects or more suitable for future clinical applications all but guarantees that an increasing number of hESC research laboratories will begin using these lines in the near future.

A.G. and A.K. contributed equally to this work.

The authors indicate no potential conflicts of interest.

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