Toward Guidelines for Research on Human Embryo Models Formed from Stem Cells

Over the past few years, a number of research groups have reported striking progress on the generation of in vitro models from mouse and human stem cells that replicate aspects of early embryonic development. Not only do these models reproduce some key cell fate decisions but, especially in the mouse system, they also mimic the spatiotemporal arrangements of embryonic and extraembryonic tissues that are required for developmental patterning and implantation in the uterus. If such models could be developed for the early human embryo, they would have great potential benefits for understanding early human development, for biomedical science, and for reducing the use of animals and human embryos in research. However, guidelines for the ethical conduct of this line of work are at present not well defined. In this Forum article, we discuss some key aspects of this emerging area of research and provide some recommendations for its ethical oversight. Over the past few years, a number of research groups have reported striking progress on the generation of in vitro models from mouse and human stem cells that replicate aspects of early embryonic development. Not only do these models reproduce some key cell fate decisions but, especially in the mouse system, they also mimic the spatiotemporal arrangements of embryonic and extraembryonic tissues that are required for developmental patterning and implantation in the uterus. If such models could be developed for the early human embryo, they would have great potential benefits for understanding early human development, for biomedical science, and for reducing the use of animals and human embryos in research. However, guidelines for the ethical conduct of this line of work are at present not well defined. In this Forum article, we discuss some key aspects of this emerging area of research and provide some recommendations for its ethical oversight. Beginning with fertilization, the mammalian conceptus (see Box 1 for terminology) goes through a characteristic developmental program with two essential outcomes: (1) the formation of extraembryonic tissues that establish connections with the maternal tissue and fuel the development of the embryo proper and (2) the generation of the three embryonic germ layers of cells that are the building blocks of the future organs. These two processes lay the groundwork for the developing embryo to form a fetus.Box 1Definition of Terms•Conceptus: the products of conception at all stages of development from zygote to birth. These include the embryo proper, the fetus, the placenta, and all extraembryonic membranes. The term “embryo proper” refers to those parts of the conceptus that will form the new body and excludes the extraembryonic tissues. Often, the terms “embryo” and “conceptus” are used interchangeably.•Pre-implantation stages of development: the first few days of development, from fertilization to implantation, during which the conceptus travels down the oviduct toward the uterus. It encompasses the 7–9 days after fertilization in humans.•Implantation: the process of attachment and invasion of the conceptus to the uterine tissues that occurs around day 7–9 after fertilization in humans. Implantation establishes the fetal-maternal interface leading to later placental development. Implantation is mediated by the polar (embryonic side near the epiblast) and mural trophectoderm cells (abembryonic side opposite the epiblast) of the blastocyst.•Post-implantation stages of development: the stages of development after the conceptus is embedded in the uterine tissues.•Gastrulation: the process by which the three germ layers of the embryonic compartment of the conceptus are formed. Gastrulation begins around day 14 in humans.•Primitive streak: the embryonic structure that establishes bilateral symmetry (alignment of equivalent structures on both sides of the anterior-posterior axis), the site of gastrulation, and the formation of the germ layers. In humans, the primitive streak appears after 14 days.•Embryonic and fetal stages: the embryonic stage begins with the division of the zygote and encompasses the development of the body plan and formation of the organs. This is followed by the fetal stage, during which growth and maturation of tissues and organs occurs. In humans, the fetal stage begins during the 9th week after fertilization and continues to birth. •Conceptus: the products of conception at all stages of development from zygote to birth. These include the embryo proper, the fetus, the placenta, and all extraembryonic membranes. The term “embryo proper” refers to those parts of the conceptus that will form the new body and excludes the extraembryonic tissues. Often, the terms “embryo” and “conceptus” are used interchangeably.•Pre-implantation stages of development: the first few days of development, from fertilization to implantation, during which the conceptus travels down the oviduct toward the uterus. It encompasses the 7–9 days after fertilization in humans.•Implantation: the process of attachment and invasion of the conceptus to the uterine tissues that occurs around day 7–9 after fertilization in humans. Implantation establishes the fetal-maternal interface leading to later placental development. Implantation is mediated by the polar (embryonic side near the epiblast) and mural trophectoderm cells (abembryonic side opposite the epiblast) of the blastocyst.•Post-implantation stages of development: the stages of development after the conceptus is embedded in the uterine tissues.•Gastrulation: the process by which the three germ layers of the embryonic compartment of the conceptus are formed. Gastrulation begins around day 14 in humans.•Primitive streak: the embryonic structure that establishes bilateral symmetry (alignment of equivalent structures on both sides of the anterior-posterior axis), the site of gastrulation, and the formation of the germ layers. In humans, the primitive streak appears after 14 days.•Embryonic and fetal stages: the embryonic stage begins with the division of the zygote and encompasses the development of the body plan and formation of the organs. This is followed by the fetal stage, during which growth and maturation of tissues and organs occurs. In humans, the fetal stage begins during the 9th week after fertilization and continues to birth. Although the mammalian conceptus can develop in vitro up to the blastocyst stage, in vivo studies are particularly challenging once implantation in the uterus occurs (approximately day 7–9 in humans), due to the small size of the conceptus and its inaccessibility in the uterine tissues. Protocols allowing for the further in vitro development of mouse blastocysts were developed starting 48 years ago (Hsu, 1971Hsu Y.C. Post-blastocyst differentiation in vitro.Nature. 1971; 231: 100-102Crossref PubMed Scopus (42) Google Scholar, Hsu, 1979Hsu Y.C. In vitro development of individually cultured whole mouse embryos from blastocyst to early somite stage.Dev. Biol. 1979; 68: 453-461Crossref PubMed Scopus (74) Google Scholar), but only recently were these applied to donated fertility clinic-derived human blastocysts (Deglincerti et al., 2016Deglincerti A. Croft G.F. Pietila L.N. Zernicka-Goetz M. Siggia E.D. Brivanlou A.H. Self-organization of the in vitro attached human embryo.Nature. 2016; 533: 251-254Crossref PubMed Scopus (310) Google Scholar). These in vitro-cultured human blastocysts reflect some aspects of early post-implantation stages (Bo et al., 2019Bo L. An Q. Zeng Q. Zhang X. Lu P. Wang Y. Zhu X. Ji Y. Fan G. Xue Z. Single-cell RNA sequencing reveals regulatory mechanism for trophoblast cell-fate divergence in human peri-implantation conceptuses.PLoS Biol. 2019; 17: e3000187Crossref PubMed Scopus (16) Google Scholar, Deglincerti et al., 2016Deglincerti A. Croft G.F. Pietila L.N. Zernicka-Goetz M. Siggia E.D. Brivanlou A.H. Self-organization of the in vitro attached human embryo.Nature. 2016; 533: 251-254Crossref PubMed Scopus (310) Google Scholar, Shahbazi et al., 2016Shahbazi M.N. Jedrusik A. Vuoristo S. Recher G. Hupalowska A. Bolton V. Fogarty N.M. Campbell A. Devito L.G. Ilic D. et al.Self-organization of the human embryo in the absence of maternal tissues.Nat. Cell Biol. 2016; 18: 700-708Crossref PubMed Scopus (296) Google Scholar), but their growth is largely confined to two dimensions, they do not yet fully mimic in vivo development, and they often show degenerative changes as culture progresses. Three-dimensional cultures of human blastocysts are now being developed that better recapitulate post-implantation development in vitro (Xiang et al., 2019Xiang L. Yin Y. Zheng Y. Ma Y. Li Y. Zhao Z. Guo J. Ai Z. Niu Y. Duan K. et al.A developmental landscape of 3D-cultured human pre-gastrulation embryos..Nature. 2019; (In press)https://doi.org/10.1038/s41586-019-1875-yCrossref Scopus (60) Google Scholar). However, the optimization of culture conditions and the extension of the development of such cultures risk contravening the internationally well-accepted 14-day rule, which limits research on intact human conceptuses/embryos (see below for definition of human embryo in law) to 14 consecutive days of development in vitro from fertilization or the appearance of the primitive streak, whichever occurs first. Given these limitations, the possibility of using human stem cells to generate in vitro models of early development represents an alternative. These models have important technical advantages, including accessibility (cell lines are more widely available than blastocysts and represent a renewable resource) and the ability to generate large numbers required for high-throughput screens and complex gene editing. A range of different and complementary models, based on several types of methodology, has been devised to mimic specific stages of mouse and human development (Figure 1) (Beccari et al., 2018Beccari L. Moris N. Girgin M. Turner D.A. Baillie-Johnson P. Cossy A.C. Lutolf M.P. Duboule D. Arias A.M. Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids.Nature. 2018; 562: 272-276Crossref PubMed Scopus (129) Google Scholar, Rivron et al., 2018bRivron N.C. Frias-Aldeguer J. Vrij E.J. Boisset J.C. Korving J. Vivie J. Truckenmuller R.K. van Oudenaarden A. van Blitterswijk C.A. Geijsen N. Blastocyst-like structures generated solely from stem cells.Nature. 2018; 557: 106-111Crossref PubMed Scopus (155) Google Scholar, Shao et al., 2017Shao Y. Taniguchi K. Townshend R.F. Miki T. Gumucio D.L. Fu J. A pluripotent stem cell-based model for post-implantation human amniotic sac development.Nat. Commun. 2017; 8: 208Crossref PubMed Scopus (101) Google Scholar, Sozen et al., 2018Sozen B. Amadei G. Cox A. Wang R. Na E. Czukiewska S. Chappell L. Voet T. Michel G. Jing N. et al.Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures.Nat. Cell Biol. 2018; 20: 979-989Crossref PubMed Scopus (106) Google Scholar, Warmflash et al., 2014Warmflash A. Sorre B. Etoc F. Siggia E.D. Brivanlou A.H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells.Nat. Methods. 2014; 11: 847-854Crossref PubMed Google Scholar). Although none of these models has yet been demonstrated to be competent to develop for more than a few days in vitro, we can envision that cell culture methodologies could be refined to a point where the models capture key features of early mammalian development with sufficient fidelity to minimize differences from the conceptus itself. When applied to human cells, such refinements will greatly enhance the power of the models but will also elevate ethical concerns over the conduct of the research. This research could result in substantial scientific and medical advances with an impact on human health. These outcomes include•attaining a fundamental understanding of key stages of early human development through the availability of versatile and scalable experimental models;•achieving a better understanding of how stem cells from different species (e.g., mouse, pig, human) and different cell lines and states vary in developmental potential, to improve their use in research and therapy;•improving assisted reproduction technologies;•understanding the biology of germ cells and infertility;•modeling implantation in three dimensions to understand the high early pregnancy loss and improve contraception technologies;•studying the genetics of early developmental defects using gene editing technologies;•revealing how defects in epigenetic reprogramming during early development can influence health throughout the lifespan, leading to strategies for disease prevention according to the developmental origins of health and disease;•refining human stem cell differentiation culture methods to achieve a greater fidelity with processes occurring during development (e.g., organogenesis). Since this field is relatively new, there is little explicit regulation of in vitro human embryo models. However, research using certain models of early human development may fall under existing legislation depending on how a human embryo is defined. Legal definitions of a human embryo vary greatly across jurisdictions and are not necessarily restricted to entities with the potential to form a full organism (Box 2). In other jurisdictions, legislation pertaining to human embryos may not include an explicit definition, leading to uncertainty around how models of human development would be viewed.Box 2Examples of Jurisdictions in which the Definition of a Human Embryo Might Capture Embryo Models Derived from Stem CellsTabled 1CountryLegal Definition of a Human EmbryoCitationAustralia“A discrete entity that has arisen from either: (a) the first mitotic division when fertilisation of a human oocyte by a human sperm is complete; or (b) any other process that initiates organised development of a biological entity with a human nuclear genome or altered human nuclear genome that has the potential to develop up to, or beyond, the stage at which the primitive streak appears; and has not yet reached 8 weeks of development since the first mitotic division.”Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006, no. 172, 2006, http://www.comlaw.gov.au/Details/C2006A00172.JapanArticle 2 (1)“(i) Embryo—A cell (except for a Germ Cell) or a cell group which has the potential to grow into an individual through the process of development in utero of a human or an animal and remains at a stage prior to placental formation.”Act on Regulation of Human Cloning Techniques (Act no. 146 of 2000), http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf.United StatesSEC. 509. “(a) None of the funds made available in this Act may be used for— (1) the creation of a human embryo or embryos for research purposes; or (2) research in which a human embryo or embryos are destroyed, discarded, or knowingly subjected to risk of injury or death greater than that allowed for research on fetuses in utero under 45 CFR 46.204(b) and section 498(b) of the Public Health Service Act (42 U.S.C. 289g(b)).(b) For purposes of this section, the term ‘human embryo or embryos’ includes any organism, not protected as a human subject under 45 CFR 46 as of the date of the enactment of this Act, that is derived by fertilization, parthenogenesis, cloning, or any other means from one or more human gametes or human diploid cells.”Dickey-Wicker Amendment, 2009, https://www.congress.gov/bill/111th-congress/house-bill/1105/text/enr.[This amendment prohibits the NIH from funding human embryo research as defined here.] Open table in a new tab Tabled 1CountryLegal Definition of a Human EmbryoCitationAustralia“A discrete entity that has arisen from either: (a) the first mitotic division when fertilisation of a human oocyte by a human sperm is complete; or (b) any other process that initiates organised development of a biological entity with a human nuclear genome or altered human nuclear genome that has the potential to develop up to, or beyond, the stage at which the primitive streak appears; and has not yet reached 8 weeks of development since the first mitotic division.”Prohibition of Human Cloning for Reproduction and the Regulation of Human Embryo Research Amendment Act 2006, no. 172, 2006, http://www.comlaw.gov.au/Details/C2006A00172.JapanArticle 2 (1)“(i) Embryo—A cell (except for a Germ Cell) or a cell group which has the potential to grow into an individual through the process of development in utero of a human or an animal and remains at a stage prior to placental formation.”Act on Regulation of Human Cloning Techniques (Act no. 146 of 2000), http://www.cas.go.jp/jp/seisaku/hourei/data/htc.pdf.United StatesSEC. 509. “(a) None of the funds made available in this Act may be used for— (1) the creation of a human embryo or embryos for research purposes; or (2) research in which a human embryo or embryos are destroyed, discarded, or knowingly subjected to risk of injury or death greater than that allowed for research on fetuses in utero under 45 CFR 46.204(b) and section 498(b) of the Public Health Service Act (42 U.S.C. 289g(b)).(b) For purposes of this section, the term ‘human embryo or embryos’ includes any organism, not protected as a human subject under 45 CFR 46 as of the date of the enactment of this Act, that is derived by fertilization, parthenogenesis, cloning, or any other means from one or more human gametes or human diploid cells.”Dickey-Wicker Amendment, 2009, https://www.congress.gov/bill/111th-congress/house-bill/1105/text/enr.[This amendment prohibits the NIH from funding human embryo research as defined here.] Open table in a new tab Moreover, this consideration of developmental or “organismic” potential (see the International Society for Stem Cell Research Box is in the the to or full human developmental potential are not under ethical (e.g., cell nuclear with species that in one specific species do not to the human the developmental of a model of one species (e.g., the is not could in the human In to uncertainty how to potential in embryo models, is also to how to the 14-day to such Stem cells derived from human blastocysts or do not have a defined developmental since cultures are not in vitro and a of developmental For human pluripotent stem cells in can form structures the early primitive streak stage 48 of the appearance of specific structures as a such as the appearance of a primitive streak, can be to and can be only after the culture is both embryos and in vitro models show in developmental and may be more or to the this uncertainty in the developmental for the of ethical such as the 14-day rule, to embryo of International Society for Stem Cell Research to Embryo Models of Research that after by an the in vitro culture of embryos or experimental generation of embryo-like structures that human potential, to of in vitro as by scientific Research vitro culture of any intact human embryo or embryo-like structure with human potential, of 14 days or formation of the primitive streak, whichever occurs human embryos or structures that human potential are utero or in any animal uterus. Research the in vitro culture of embryos or experimental generation of embryo-like structures that human potential, to of in vitro as by scientific vitro culture of any intact human embryo or embryo-like structure with human potential, of 14 days or formation of the primitive streak, whichever occurs human embryos or structures that human potential are utero or in any animal uterus. there is little in the of specific guidelines to provide for proper ethical of this the substantial scientific and medical to be et al., N. M. J. Arias A. Zernicka-Goetz M. Fu J. van S. A. G. et of embryo models from stem cells.Nature. 2018; PubMed Scopus (42) Google Scholar). It is that any on the future of this research be and on a this we to some ethical to different embryo models and some guidelines for the conduct of this research. that do not to model the development of the conceptus are not equivalent to This includes research that embryonic or extraembryonic cell (e.g., or extraembryonic or of embryonic and extraembryonic that are not to the development of an intact embryo (e.g., sac In these we can from experimental studies in that such are not to develop into embryos or fetuses further with all extra-embryonic tissues. sac and cultures can provide models equivalent ethical that to model the development of the conceptus to be extraembryonic the in vivo development of the human conceptus the trophectoderm of the blastocyst the process of in utero implantation, embryo models that cells the and extraembryonic and by cells (e.g., do the possibility of a with further potential to the human uterus. research in that the developmental potential of such models is from certain at this of research on human embryo models that do to model the development of the conceptus be by the to regulation of embryo research. on the research is under on lines of human stem cells, on parts of and on embryos developed up to 14 for of such research in jurisdictions, Embryo Research in the Human and Embryo in the Stem Cell in and Embryo Research in of this research through funding by and is to proper scientific and ethical into the of the research. Human embryo models not be for use in assisted reproduction at a pregnancy but could be used as in vitro models to develop to the of human both the potential biomedical benefits of the work and its scientific and into the ethical and benefits of human embryo with available stem cell However, the scientific and of the research to be that model development and post-implantation development up to by human embryonic and extraembryonic including the trophoblast and extraembryonic with the to represent the development of the conceptus up to the appearance of the primitive streak or are only by in the or equivalent more where that do not model the of all embryonic and extraembryonic or models that the potential to form a full organism are from but are to the or equivalent and subject to the body that model human and stages the appearance of the primitive are from they do not all of the conceptus (embryonic and in an intact and are at a discrete and defined of development or discrete of than the development of an intact embryo or models of the or stem cell cultures three germ or research would be to the or equivalent and subject to the body human embryo model that at the of appearance of the primitive streak into parts for further culture or in vitro would be subject to the in (1) and may be to be from further in vitro of human embryo models with animal or human cells or tissues or embryos be subject to the in (1) and (2) and This would include in vitro embryo model embryo embryo or any of these in vitro into uterine tissues or uterine human embryo model in any of the be in vivo into the uterus of an animal or Research in this area is and the of this research by and is to proper scientific and ethical oversight. An based process to that a regulatory can be be and into the potential benefits of this research and the power of these models. regulatory is to legislation that could have in the The is in a to recommendations for the conduct of this as has in the past for other challenging its The guidelines for Stem Cell Research and Box provide some recommendations to this research but to be and in the of the progress in this recommendations as a starting point for the Society and the at the to this work and are in is by the a

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