Review Article https://doi.org/10.1038/s41563-020-00829-9

Stem-cell-based embryo models for fundamental research and translation

Jianping Fu 1,2,3 ✉ , Aryeh Warmflash 4,5 ✉ and Matthias P. Lutolf 6,7 ✉

Despite its importance, understanding the early phases of human development has been limited by availability of human sam- ples. The recent emergence of stem-cell-derived embryo models, a new field aiming to use stem cells to construct in vitro models to recapitulate snapshots of the development of the mammalian conceptus, opens up exciting opportunities to promote fundamental understanding of human development and advance reproductive and regenerative medicine. This Review provides a summary of the current knowledge of early mammalian development, using mouse and human conceptuses as models, and emphasizes their similarities and critical differences. We then highlight existing embryo models that mimic different aspects of mouse and human development. We further discuss bioengineering tools used for controlling multicellular interactions and self-organization critical for the development of these models. We conclude with a discussion of the important next steps and exciting future opportunities of stem-cell-derived embryo models for fundamental discovery and translation.

he development of a multicellular organism from a single notable progress in studying non-human primate (NHP) monkey fertilized egg is a brilliant triumph of evolution that has fas- embryos10–12, whose developments are similar to humans. However, Tcinated generations of scientists (Box 1). Understanding our NHP monkey models remain costly, are difficult to modify geneti- own development is of particular fundamental and practical inter- cally, and have their own ethical challenges. est; however, it poses a unique set of technical and ethical challenges. Recent advances in mammalian embryology, stem cell biol- Our current knowledge of embryonic development is derived from ogy, organoid technology, and bioengineering have contributed a number of animal species, chosen because they are convenient to to a considerable interest in the development of multicellular study and amenable to experimental manipulation or genetic analy- systems based on emergent self-organization and tissue pattern- sis1. These studies have revealed developmental principles (Box 2), ing. Importantly, different models of the mammalian conceptus and signalling and transcriptional networks that underlie cell fate have been developed using mouse and human stem cells13–30. This patterning and tissue morphogenesis. In particular, most of our emerging field aims to use stem cell cultures to create organized knowledge of mammalian development derives from the mouse embryo-like structures (or embryoids), whose development and model. However, it is becoming evident that there are morphologi- architecture bear remarkable similarities to their in vivo coun- cal and genetic differences between mice and humans that make terparts. Embryoids are distinct from organoids, as organoids cross-species comparisons problematic2. are organized multicellular structures that mimic the develop- Knowledge of human embryogenesis, which is critical for ment, regeneration and homeostasis of a single tissue or organ. assisted reproductive technologies and prevention of pregnancy In contrast, embryoids aim to model integrated development of loss, birth defects and teratogenesis (Box 3), should ideally be the entire conceptus or a considerable portion thereof. In gen- learned from studying the human embryo per se; however, such eral, embryoids have a more reproducible cellular organization studies have been challenging due to limited access to and bioethical and architecture than organoids, as bioengineering approaches constraints on human embryo specimens. Excess pre-implantation are commonly deployed to guide their development and culture human embryos generated in in vitro fertilization (IVF) clinics are time is limited to a few days. In addition, the stem cells used in available for research3,4; however, once a human embryo implants embryoids have been well established and their cultures are usu- into the uterus, subsequent development is hidden from direct ally robust. This Review discusses the developmental principles observation. Recent progress in prolonged in vitro culture of IVF manifested in the development of embryoids and their appli- human embryos has opened the door for genetic and molecu- cations for advancing human embryology (particularly at the lar studies of human embryos directly5–7. However, international post-implantation stages) and reproductive and regenerative guidelines prohibit in vitro culture of human embryos beyond 14 medicine. Bioengineering tools used for controlling multicellular days post-fertilization (embryonic day 14, E14.0) or reaching the interactions and self-organization critical for embryoid develop- onset of primitive streak (PS) development (‘the 14-day rule’)8,9, ment are highlighted. We conclude with a discussion of impor- which marks the outset of gastrulation. The bioethical regulation of tant next steps to leverage advanced bioengineering controls of human embryo culture has limited studies of IVF human embryos multicellular interactions to promote the continuous, progressive for understanding post-implantation human development. There is development of this exciting nascent field.

1Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA. 2Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA. 3Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA. 4Department of Biosciences, Rice University, Houston, TX, USA. 5Department of Bioengineering, Rice University, Houston, TX, USA. 6Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences and School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. 7Institute of Chemical Sciences and Engineering, School of Basic Science, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. ✉e-mail: [email protected]; [email protected]; [email protected]

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Box 1 | Glossary Box 2 | Classic developmental concepts and principles

Conceptus. Te products of conception at all stages of develop- Developmental potency. Developmental potency describes the ment from zygote to birth. Tese include the embryo proper, the ability of a cell in the embryo to diferentiate into other cell types. fetus, the placenta and all extraembryonic membranes. Te term Tere is a continuum of developmental potency following pro- ‘embryo proper’ refers to those parts of the conceptus that will gressive development of the embryo, from totipotency, pluripo- form the new body and excludes extraembryonic tissues. Ofen, tency, multipotency, oligopotency and fnally unipotency. the terms ‘embryo’ and ‘conceptus’ are used interchangeably. Pattern formation. Pattern formation is the process by which Pre-implantation development. Te frst few days of initially equivalent cells in a developing tissue acquire identities development, from fertilization to implantation, during which that lead to a well-ordered spatial pattern of cell activities. Pattern the conceptus travels down the oviduct toward the uterus. It formation can involve positional information or cell sorting (see encompasses the frst 7–9 days afer fertilization in humans. below). Morula. Te very early stage in a conceptus when cleavage has Positional information. Te concept of positional information resulted in a solid ball of cells. proposes that cells in a developing tissue acquire positional Implantation. Te process of attachment and invasion of the values as in a coordinate system, which they interpret by conceptus to the uterine tissues that occurs around day 7–9 developing in particular ways to give rise to spatial patterns. afer fertilization in humans. Implantation establishes the fetal– Positional information ofen involves difusion of morphogens, maternal interface leading to later placental development. leading to signalling gradients and diferential gene expression in a morphogen concentration-dependent manner. A key feature Blastula and blastocyst. Te stage of the conceptus prior to of positional information being the basis for pattern formation is implantation is termed blastula. At this stage, the conceptus is that there is no pre-pattern in the developing tissue. called a blastocyst. Cell sorting. Pattern formation in a developing tissue can initiate Peri-implantation development. Te development of the from the specifcation of diferent cell types in the tissue in a conceptus in the uterine tissues prior to gastrulation. salt-and-pepper fashion, which is followed by sorting of diferent Gastrula and gastrulation. Gastrulation describes the process cell types into distinct domains from where diferent tissues are by which the three defnitive germ layers of the embryonic formed. Cell sorting involves a morphogenetic process during compartment of the conceptus are formed. Gastrulation begins which individual cells exchange neighbours, increasing the around day 14 in humans. Te gastrulation stage conceptus is number of homotypic contacts and decreasing the number of termed gastrula. heterotypic contacts. Primitive streak. Te embryonic structure that breaks radial Symmetry breaking. Symmetry breaking is the process by symmetry by establishing the anterior–posterior axis and which an initially homogeneous system acquires an asymmetry establishes bilateral symmetry (alignment of equivalent along an axis. While external cues can induce or assist symmetry structures on both sides of the anterior–posterior axis), the site breaking, asymmetries can emerge spontaneously without such of gastrulation, and the formation of the germ layers. In the input, guided by self-organization (see below). human embryo, the primitive streak appears around day 14 afer Self-organization. Self-organization, as a non-equilibrium fertilization. process, can be defned as the formation of complex patterned Neurula and neurulation. Neurulation describes the process structures from units of less complexity by collective, nonlinear by which the neural tube is formed from the ectodermal neural interactions, without referring to an external blueprint or plate. Te neural tube will give rise to the brain and spinal cord. template. Tese local internal interactions typically form Te neurulation stage conceptus is termed neurula. feedback loops, thereby conferring robustness to the system. Organogenesis. Te development of specifc organs in the Other common features found in self-organizing systems are embryo such as the brain and heart. Organogenesis starts soon nonlinearity, symmetry breaking, and the emergence of patterns afer gastrulation. In humans, organogenesis commences during through amplifcations of stochastic fuctuations. the fourth week afer fertilization. Embryonic induction. Embryonic induction is an interaction Embryonic and fetal stages. Te embryonic stage begins with between one (inducing) tissue and another (responding) tissue, the division of the zygote and encompasses the development of as a result of which the responding tissue undergoes a change in the body plan and formation of the organs. Tis is followed by its direction of diferentiation. the fetal stage, during which growth and maturation of tissues Signalling centre. A localized region of the embryo that exerts and organs occurs. In humans, the fetal stage begins during the a special infuence on surrounding cells, usually by means of ninth week afer fertilization and continues to birth. secreted signalling molecules, and thus instructs how those cells develop. Mammalian development as a reference framework Organizer. A signalling centre that directs the development of The development of embryoids, like that of embryos, involves the the whole embryo or of part of the embryo. emergence of organized multicellular structures, through coor- Community efect. Community efect describes cell–cell dinated cellular processes including pattern formation, morpho- communication among a group of nearby cells in a developing genesis, cell differentiation and growth. Here we first discuss the tissue, which is necessary for them to maintain coordinated principles of early mouse and human development before turning behaviours. to how these are manifested in embryoids. Morphogenetic regulation. Tissue-scale morphogenetic During early development, both mouse and human embryos changes, including changes in cell shape, number, position and develop from a zygote and proceed through recognizable stages of force, can work in concert with classic developmental signalling morula, blastula, gastrula, neurula and organogenesis (Fig. 1, Box events mediated by difusible signals to regulate gene expression 1, Box 4). The overall programme of pre-implantation development and cell fate specifcation. from a zygote to a blastocyst is conserved between mice and

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Box 3 | Clinical benefts of human embryology Box 4). Mouse development from E5.0–E6.5 leads to the formation of a cup-shaped EPI juxtaposed with TE-derived extraembryonic ectoderm (ExE), enclosing the pro-amniotic cavity. Concurrently, Advancing fundamental understanding of human embryogen- the PE forms the visceral endoderm (VE) that envelops both esis can provide a scientifc foundation for improving assisted EPI and ExE. In contrast, soon after human blastocyst implanta- human reproduction and prevention of pregnancy loss, birth de- tion, while the EPI undergoes epithelization and lumenogenesis fects and teratogenesis. It will also advance the biology of germ to form the pro-amniotic cavity5–7, EPI cells closer to invading TE cells and treatment of infertility. Advancing understanding of cells become specified into the amniotic ectoderm (AM)34, with human implantation will help develop efective contraception remaining pluripotent EPI cells forming the embryonic disc. Thus, technologies and treatments of recurrent implantation failure. in pre-gastrulation human embryo, the pro-amniotic cavity is sur- Detailed understanding of the widespread epigenetic program- rounded by a continuous epithelium with AM cells on one side and ming during human embryonic development can provide im- EPI cells on the other. portant insights for disease progression in later life. Studying Studies of mouse gastrulation support the importance of extra- human development is critical for improving stem cell diferen- embryonic tissues35,36 (Fig. 1, Box 4). In particular, regional pattern- tiation protocols to mimic embryogenesis, to achieve desired cell ing of VE in pre-gastrulation mouse embryo leads to a gradient of functions for research and therapy. WNT and NODAL signalling and the establishment of the anterior– posterior (A–P) axis of the embryo37,38. Importantly, developmental signals involving WNT, NODAL and BMP at the proximal, poste- humans, leading to the formation of the blastocyst, containing an rior end of the EPI instruct EPI cells to form the PS by E6.5 and outer trophectoderm (TE) layer surrounding a cavity (blastocoel) ingress through the PS to acquire mesoderm and endoderm fates39– and an inner cell mass (ICM) on one side of the cavity31,32 (Fig. 1, 41. Human gastrulation initiates around E14.0. However, given Box 4). As the blastocyst develops, the ICM becomes further segre- limited access to post-implantation human tissues, gastrulation gated into two cell populations: the pluripotent epiblast (EPI) and remains one of the most mysterious phases of human development. primitive endoderm (PE; or hypoblast in human)31,32. During mouse gastrulation, primordial germ cells (PGCs), pre- The timing of blastocyst implantation differs between mice and cursors of sperm and egg, emerge at the boundary between pos- humans (E5.0 in mouse and E7.0 in human). Furthermore, morpho- terior EPI and ExE42,43. Data on human PGC specification remain genesis and lineage developments during early post-implantation sparse44. Existing data from NHP monkey embryos45 suggest that mouse and human development show distinct features2,33 (Fig. 1, primate PGCs may emerge in the nascent AM prior to the gastrula-

Mouse E3.0 E3.5 E4.5 E5.5 E6.5

Early Mature Zygote Morula blastocyst blastocyst

Post-implantation Gastrulation embryo

Human E6.0 E7.0 E10.0 E14.0

Fig. 1 | Overview of mouse and human development from pre-implantation to the onset of gastrulation. Prior to implantation, both mouse and human embryos undergo cell divisions culminating in the development of a blastocyst, comprising an outer trophectoderm (TE) layer and an inner cell mass (ICM) that further segregates into epiblast (EPI, light blue) and primitive endoderm (PE, yellow; hypoblast in humans). The timing of blastocyst implantation differs between mice and humans (E5.0 in mouse and E7.0 in human). Furthermore, morphogenesis and lineage developments during peri-implantation mouse and human development show distinct features. Mouse development from E5.0–E6.5 leads to the formation of a cup-shaped EPI juxtaposed with TE-derived extraembryonic ectoderm (ExE), enclosing the pro-amniotic cavity. Concurrently, the PE forms the visceral endoderm (VE) that envelops both EPI and ExE. In contrast, soon after human blastocyst implantation, while the EPI undergoes lumenogenesis to form the pro-amniotic cavity, EPI cells adjacent to polar TE cells become specified into the amniotic ectoderm (AM, dark blue), with remaining pluripotent EPI cells forming the embryonic disc. By E6.5 for mice and E14.0 for humans, gastrulation is initiated in the posterior EPI compartment (gastrulating cells in red). Mouse primordial germ cells (PGCs) emerge at the boundary between posterior EPI and ExE at the onset of gastrulation (not shown). Data on primate PGC specification remain sparse. Existing data suggest that human PGCs may emerge in the nascent AM prior to the gastrulation (not shown). Human PGC specification requires additional studies for clarification.

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Box 4 | Early mouse and human development

Pre-implantation development. Pre-implantation mouse and Soon afer, gastrulation is initiated at the proximal, posterior end human development displays intricate self-organization and au- of the EPI by a convergence of BMP-WNT-NODAL signalling tonomy. Afer fertilization, the one-cell zygote undergoes cleavage interactions between EPI, ExE and VE39–41. Te antagonists cell divisions to form a solid ball of cells resembling a mulberry secreted by the AVE block signalling and impart neuroectoderm (and hence the name morula). Cells of the morula begin to difer- characters at the anterior pole of the EPI38, whereas signals at the entiate, leading to blastocyst formation. In the blastocyst, the tro- proximal, posterior end of the EPI instruct cells to undergo the phectoderm (TE) surrounds a fuid-flled cavity (blastocoel) with epithelial–mesenchymal transition (EMT) and ingress through the an inner cell mass (ICM) on one side. Te TE is an extraembryonic PS to acquire mesendodermal fates39. During mouse gastrulation, tissue and will give rise to the placenta. As the blastocyst develops, primordial germ cells (PGCs) emerge at the proximal, posterior the ICM becomes segregated into two distinct cell populations: end of the EPI42,43. Experimental evidence supports that the embryonic epiblast (EPI), which will give rise to the embryo prospective PGCs are selected from somatic, gastrulating EPI cells proper, and a second extraembryonic tissue known as the primi- by dose-dependent BMP signals that originate from the ExE114. tive endoderm (PE; or hypoblast in human). Pre-implantation de- Gastrulation remains one of the most mysterious phases of velopment has been extensively studied using the mouse embryo, human development. Recent studies of NHP monkey embryos revealing important cellular and morphogenetic events including suggest conserved mechanisms are likely to be in play for A–P position-dependent TE/ICM patterning, the blastocoelic cavity patterning during human gastrulation12,45. Limited data from NHP formation, and lineage segregation and sorting of EPI and PE in monkey45 and in vitro cultured human embryos44 suggest that PGCs the ICM. Te readers are referred to some excellent reviews on the may emerge in the dorsal nascent AM soon afer implantation. mouse blastocyst development31,32. Human blastocyst formation Tis unexpected fnding will require additional confrmation. remains incompletely understood3,111. Existing data suggests that Afer gastrulation, the EPI in mouse and human embryos there are diferences in timing, in gene expression and potential- transforms into a trilaminar structure consisting of ectoderm, ly in mechanisms of lineage development and function between mesoderm and endoderm. As gastrulation proceeds, it brings mice and humans during pre-implantation development2. subpopulations of cells in the three germ layer linages into proximity so that they can undergo inductive interactions to Peri-implantation development. Successful implantation involves pattern layers and specify new cell types, driving the development a bilateral interaction between a competent blastocyst and a of organ rudiments. receptive uterus. Implantation of the blastocyst (E5.0 in mouse and E7.0 in human) triggers major morphological reorganization and Neurulation. Gastrulation is followed by neurulation. During lineage developments. Upon implantation of the mouse blastocyst, neurulation, the ectoderm is frst patterned into the neuroectoderm the TE adjacent to the EPI (polar TE) forms the extraembryonic (neural plate) in the medial portion of the embryo fanked by ectoderm (ExE) and ectoplacental cone. Concomitantly, the EPI future epidermis. At the border between these regions, the neural and ExE each undergo lumenogenesis so that separate apical crest and placodes form in response to BMP and WNT signals lumens are formed in each compartment84,105. Te two lumenal emanated from surrounding tissues115,116. Te neural plate soon cavities soon fuse to establish the pro-amniotic cavity, leading to folds into the neural tube (NT)46,47, with its anterior and posterior the formation of a cup-shaped EPI juxtaposed with the ExE at E6.0 regions giving rise to the brain and spinal cord, respectively. Cells (the egg cylinder). From E5.0 to E6.0, the PE forms the parietal in the NT continue to diferentiate under the infuence of inductive endoderm and visceral endoderm (VE). By E6.0, the VE envelops factors emanating from surrounding tissues. Sonic hedgehog both the EPI and ExE, setting the stage for gastrulation. (Shh)-mediated transcriptional networks that control ventral Morphogenesis and lineage development in the peri- patterning of the mouse spinal cord have been well elucidated48,49. implantation human embryo show distinct features compared Human neurulation remains challenging to study, even with mice34. Upon implantation, the EPI undergoes lumenogenesis though NT closure defects remain one of the most common to form the pro-amniotic cavity5–7, similar to the mouse EPI. birth defects46,47. Recent studies suggest that late-manifesting Distinctly, the lumenal EPI soon breaks symmetry and resolves neurodegenerative disorders, such as Huntington’s disease, may into the bipolar patterned EPI-amnion sac7,33. Specifcally, EPI have a neurodevelopmental component25,117. Te role of early cells adjacent to invading polar TE cells diferentiate into the nervous system development in late-onset neurodegenerative amniotic ectoderm (AM)7,33, an extraembryonic tissue involved in disorders remains a debated topic. future fetal membrane development. Te EPI cells at the opposite While the ectoderm undergoes neurulation, the mesoderm and pole adjacent to the hypoblast remain pluripotent and become endoderm also become further specifed. Specifcally, mesodermal thickened and more columnar, forming the embryonic disc. Tus, cells are organized into cardiogenic mesoderm, axial mesoderm the pre-gastrulation EPI displays distinct topologies between of the prechordal plate and notochord, paraxial mesoderm, humans and mice: discoid in human and cup-shaped in mouse. intermediate mesoderm and lateral plate mesoderm. Each of these Te mouse embryo does not develop the bipolar EPI-amnion mesodermal regions undergoes some form of segmentation. Te structure. In mice, the AM emerges as amniotic folds at the most evident and complete segmentation occurs in the paraxial junction of the EPI and ExE during gastrulation112,113. trunk mesoderm, where each segment becomes an entirely Gastrulation. Prior to mouse gastrulation, reciprocal separate somite. Much of the other mesodermal regions develop interactions between EPI, ExE and VE lead to regionalized into embryonic connective tissues, cardiovascular and lymphatic patterning in these tissues35,36. Regionalization of VE is particularly systems, skeletal muscle cells, most of the urogenital system, important, leading to the formation of the anterior VE or AVE at and the lining of the pericardial, pleural and peritoneal cavities. the prospective anterior side of the embryo37,38. Te AVE secrets Following gastrulation, the endoderm folds to form the primitive antagonists to shield overlying EPI cells from diferentiation. gut tube consisting of three subdivisions: foregut, midgut and Development of the AVE thus breaks radial symmetry and marks hindgut, which subsequently give rise to the epithelial lining of anterior–posterior (A–P) axis formation in the mouse embryo. the digestive and respiratory systems.

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Naive ESCs Induced embryonic and extraembryonic stem cells

EPSCs EpiLCs EpiSCs

XEN cells

TSCs Mouse

E2.0 E4.5 E5.75 E7.0ÐE8.0 Adult E5.0 E7.0 E12.0ÐE13.0

Human TSCs

HypoSCs

EPSCs Primed ESCs

Induced embryonic and extraembryonic Naive ESCs stem cells

Fig. 2 | Mouse and human embryonic and extraembryonic stem cells and their corresponding developmental potencies. Expanded potential stem cells (EPSCs) are established by isolating individual cells (or blastomeres) from eight-cell stage mouse and human embryos (or morula). By isolating cells from mouse blastocysts, mouse embryonic stem cells (mESCs) with naive pluripotency, trophoblast stem cells (mTSCs), and extraembryonic endoderm (mXEN) cells representing the stem cell population of the primitive endoderm (PE) have been established. Similarly, human trophoblast stem cells (hTSCs) have been derived from human blastocyst. Primed mouse ESCs, known as mouse epiblast stem cells (mEpiSCs), are derived from the late post-implantation, pre-gastrulation mouse EPI. Mouse EPI-like cells (EpiLCs) with an intermediate or formative state between naive and primed pluripotency have been generated from mESCs in vitro, with a transcriptional profile similar to the early post-implantation mouse EPI. Human ESCs (hESCs) with primed pluripotency have also been derived from pre-implantation human blastocysts. Using strategies such as reprogramming, differentiated somatic mouse and human cells can be converted to induced embryonic and extraembryonic stem cells, including induced pluripotent stem cells, or iPSCs. Using chemical cocktails, primed hESCs can be reverted into a naive-like pluripotent state. Human hypoblast stem cells (hypoSCs) can be generated using these chemically reset naive hESC lines. tion. Additional studies are required to determine whether the same which they readily differentiate50. The transition between naive and is true for human PGCs. primed states has been referred to as formative pluripotency during In mouse and human embryos, gastrulation transforms the which EPI cells gain the capacity to make lineage decisions51. EPI into a trilaminar structure consisting of definitive ectoderm, Mouse pluripotent stem cells (mPSCs) in culture display a simi- mesoderm and endoderm. The three germ layers undergo induc- lar continuum of states. Mouse embryonic stem cells (mESCs) with tive interactions to pattern layers and specify new cell types, driving naive pluripotency can be isolated directly from the ICM of the organ rudiment development (Box 4). Following gastrulation, the mouse blastocyst and maintained in culture52,53. In contrast, mouse ectoderm undergoes neurulation, in which the neural plate is first stem cells corresponding to the primed state, known as mouse epi- patterned in the dorsal ectoderm before folding into the neural tube blast stem cells (mEpiSCs), are derived from the post-implantation (NT)46,47 (Box 4). Cells in the NT continue to differentiate into dif- mouse EPI54,55. mEpiSCs exhibit more advanced developmental fea- ferent classes of neuronal progenitors48,49. Concomitantly, mesoder- tures consistent with the early-gastrulation EPI56. Mouse EPI-like mal cells are organized into different regions to from the primordia cells (mEpiLCs) with formative pluripotency have been generated of major organ systems including cardiovascular and lymphatic sys- from mESCs in vitro, with a transcriptional profile consistent with tems and skeletal muscle cells. Simultaneously, the endoderm will early post-implantation mouse EPI57. fold to form the primitive gut tube, which will produce the digestive Stem cell lines representative of extraembryonic lineages in and respiratory systems. the mouse blastocyst have also been established, including mouse trophoblast stem cells (mTSCs)58 and extraembryonic endoderm Embryonic and extraembryonic stem cells as building (XEN) cells representing the stem cell population of the PE59. blocks As a bottom-up approach, the development of embryoids uses Human stem cells. Human ESCs (hESCs) have also been success- embryonic and extraembryonic stem cells, including those derived fully derived from human blastocysts60. However, hESCs have a from embryos, as building blocks to construct models to recapitu- transcriptome and methylome different from the EPI of the human late embryonic development (Fig. 2). blastocyst61,62, suggesting that the conditions in which hESCs are cultured fail to capture the pre-implantation developmental pro- Mouse stem cells. The EPI cells of the mouse blastocyst are plu- gramme of the human embryo. Instead, hESCs are developmentally ripotent, and their functional, epigenetic and signalling properties similar to the post-implantation, pre-gastrulation EPI in cynomol- have been extensively characterized. These studies reveal that plu- gus monkey embryos12. Consistently, hESCs more closely resemble ripotency is dynamic and progressive. As the mouse embryo devel- mEpiSCs than mESCs in terms of molecular properties, lineage ops from blastula to gastrula, EPI cells transit from a naive state, in potency and culture conditions50,54,55. However, there are still some which they do not respond to inductive signals, to a primed state in notable differences in gene expression63 and in the propensity for

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PGC formation between hESCs and mEpiSCs, as hESCs can initi- inefficient. Optimization studies yielded improved conditions in ate PGC formation whereas mEpiSCs cannot64,65. There are recent which the relative proportions of the three cell lineages (EPI-, TE- reports showing derivations of hESC lines from human blasto- and PE-like cells) more closely resemble that of mouse blastocyst81. cysts with naive pluripotency features66,67, and of chemical repro- Remarkably, culture of mEPSCs in appropriate conditions yields gramming cocktails capable of converting hESCs from primed to self-organized blastoids consisting of EPI-, TE- and PE-like cells30. naive pluripotency68,69. However, functional validations of naive Another recent work mixing mEPSCs with mTSCs also led to the pluripotency including chimera formation and germline transmis- formation of blastoids that showed developmental progression from sion as well as tetraploid complementation, which have been used the pre- to post-implantation egg cylinder morphology in vitro82, for mESCs, cannot be implemented with human cells for ethical similar to the mouse ETX embryoid described below. Although reasons. To address this issue, there are ongoing discussions of a capable of implanting in the mouse uterus, all of the mouse blas- testable functional framework to assess naive pluripotency in toids fail to develop much further than the blastocyst stage either human cells33. in vitro or in vivo. The reasons for this are not currently under- Somatic human cells can also be converted to a pluripotent stood and may point to a requirement for greater organization than state by cell fusion, somatic cell nuclear transfer, transcription currently achieved in mouse blastoids. It remains to be determined factor-based reprogramming, and chemical reprogramming70. whether human blastoids can be generated by using either naive Pluripotent stem cells generated by these reprogramming strategies hPSCs or hEPSCs with or without hTSCs or hypoSCs under suitable are called induced pluripotent stem cells, or iPSCs. Human iPSCs culture environments. (hiPSCs) are considered molecularly and functionally equivalent to hESCs71. Human amniotic sac embryoid. During early post-implantation Recently, through chemical screening, individual blastomeres human development, a patterned bipolar EPI-AM structure arises isolated from eight-cell stage mouse morula were successfully cul- from the EPI. It was recently shown that culturing primed hPSCs tured to establish mouse expanded potential stem cells (mEPSCs) on a soft culture surface together with native extracellular matrix that appear to possess developmental potency for all embryonic and (ECM) proteins (that is, Geltrex) diluted in culture medium leads to extraembryonic lineages in blastocyst chimaera assays72,73. Using the formation of a spherical lumenal hPSC colony20. This observa- similar approaches, human EPSCs (hEPSCs) were also derived tion is consistent with the intrinsic lumenogenic property associ- from primed hESCs and hiPSCs, and hEPSCs are shown to have the ated with primed but not naive hPSCs83,84. Interestingly, hPSCs in potency to form trophoblast stem cells74. the colony lose pluripotency and differentiate into amniotic cells, In contrast to mouse extraembryonic stem cells, human extra- even without exogenous inductive factors in the culture medium20. embryonic stem cells have only emerged recently. Using chemical If only one of these culture elements is present, either a soft substrate screening, human trophoblast stem cells (hTSCs) were first derived or diluted gel in the culture medium, primed hPSCs form lumenal from human blastocysts and first-trimester placental tissues75. sacs but retain pluripotency, suggesting that amniotic differentia- Human hypoblast stem cells (hypoSCs) were also recently reported tion of hPSCs is mechanosensitive20. Amniotic differentiation of using chemically reset naive hESC lines76. Recent work showed that hPSCs requires endogenous BMP signalling, and its inhibition chemically reprogrammed naive hESCs could give rise to hTSCs under amnion-differentiation conditions is sufficient for rescuing when cultured in appropriate conditions77, and that both chemi- hPSC pluripotency20. cally reset and embryo-derived naive hESCs could be used to derive A small fraction of lumenal sacs, rather than differentiating hTSC lines78. With the emergence of these human extraembryonic entirely into amniotic tissues, spontaneously break symmetry and cell lines as well as hEPSCs, it becomes imperative for additional form a bipolar structure with columnar pluripotent cells on one molecular and functional characterizations for authentication side and amniotic cells on the other, mimicking EPI-AM pattern- and establishing their developmental identities compared to their ing in the pre-gastrulation human embryo20. This model is termed in vivo counterparts78,79. the post-implantation amniotic sac embryoid (PASE). Symmetry breaking in the PASE also depends on BMP activity, and active A rapidly growing toolbox of embryoids BMP signalling is only evident at the prospective AM-like pole20. Stem cells serve as building blocks for the development of embry- Progressive development of the PASE results in EPI-like cells fur- oids that recapitulate different stages of mammalian development, ther differentiating into PS-like cells. This spontaneous symmetry from blastula through gastrula or early neurula and organogenesis breaking occurs in only 5–10% of lumenal hPSC sacs. To increase (Fig. 3). Development of embryoids use the same developmental efficiency of PASE formation, a microfluidic PASE model has principles that manifest in mammalian development. Importantly, recently been developed27. This microfluidic device allows small embryoids have already generated new insights into early mamma- clusters of primed hPSCs to be grown in small indentations with lian development. separate channels supplying culture medium to each side27. Flowing BMP4 in only one of these channels leads to reproducible pat- Embryoid to model blastocyst development. The first embry- terning of amniotic cells only on the side exposed to BMP427. The oid to model blastocyst formation (or blastoid) was successfully opposite side remains pluripotent but soon goes on to differentiate developed by mixing mESCs with mTSCs at a defined ratio under into PS-like cells27. The identity of PS derivatives can be modulated appropriate culture conditions, leading to their self-assembly into by stimulating this side of the cell clusters with additional ligands: a tissue organization reminiscent of the mouse blastocyst22. As WNT stimulation together with either BMP4 or ACTIVIN-A leads in vivo, mouse blastoids possess an outer TE layer surrounding a to posterior and anterior PS derivatives, respectively27. Importantly, compact ICM-like compartment, and their transcriptome is more human PGC-like cells (hPGCLCs) emerge in the microfluidic PASE similar to mouse blastocyst than is achieved by simply combining before it initiates gastrulation-like events27, suggesting applications mESC and mTSC transcriptomes. Mouse blastoids have been used of the microfluidic PASE model for studying the origin and specifi- to dissect interactions between embryonic and extraembryonic cation of hPGCs. compartments, revealing that NODAL and BMP signals from the The PASE represents the first embryoid to model early ICM-like compartment are important for growth and morphogen- post-implantation development of the EPI and AM compartments esis of TE cells22. This insight has proven useful for improving cul- of the human embryo. It also suggests the inductive role of AM in ture conditions of mTSCs80. In the initial blastoid protocol, further triggering human gastrulation. Although the microfluidic PASE cell segregation and sorting of ICM-like cells into PE-like cells was model considerably improves the controllability of EPI-AM pat-

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E4.5 E5.5 E6.5

Implantation Gastrulation

3D blastoid

3D ETX embryoid

3D gastruloid

3D trunk-like structure

2D gastrulation model

E7.0 E10.0 E14.0

Implantation Gastrulation

3D epiblast patterning model

3D PASE

2D gastrulation model

3D gastruloid

2D neurulation model

Fig. 3 | Existing embryoids that recapitulate different stages of mouse and human development. 3D blastoids are embryoids for modelling pre-implantation blastocyst development. 3D ETX embryoids model post-implantation embryo development up to early gastrulation. 3D gastruloid and trunk-like structure are embryoids to model post-gastrulation development of the posterior portion of the embryo. The 2D gastrulation model is an embryoid for modelling germ layer patterning during gastrulation. The 3D epiblast patterning model is an embryoid for modelling epiblast morphogenesis and patterning during early post-implantation development. 3D post-implantation amniotic sac embryoid (PASE) models post-implantation human development up to early gastrulation. 2D neurulation models are embryoids for modelling the neurulation process, leading to neural tube development and patterning. terning, further asymmetries are not demonstrated and the EPI-like live imaging of the 2D gastrulation model has allowed quantifica- compartment is either entirely anterior or posterior in character. tion of the self-organized signalling dynamics that drive these pat- Prolonged culture of the PASE is also limited by the confined space terning events87–90. These studies have revealed that rather than in the microfluidic device. Furthermore, disseminating cells from creating stable gradients, cells generate dynamic expanding fronts the PASE mimicking gastrulation would lead to its disassembly. of endogenous WNT and NODAL signalling that are interpreted Future efforts should be devoted to identifying a strategy to prolong combinatorially to pattern different germ layers. These studies can the culture of the PASE and promote self-organization of gastrulat- serve as a template for investigating the mechanisms of patterning ing cells. It is possible that adding human extraembryonic stem cells through signalling dynamics in other embryoids. Interestingly, in including hypoSCs to the PASE will be helpful for these efforts. colonies treated with WNT rather than BMP4, similar cell fate pat- terns are observed but with a different mechanism, which involves Two-dimensional models of gastrulation. Treatment of primed a wave of epithelial–mesenchymal transition (EMT) that sensi- hPSCs confined to micropatterned colonies with BMP4 reproducibly tizes cells to the exogenous WNT signal91. Mouse gastrulation has leads to organized differentiation with putative TE-like cells on the also been successfully modelled using the 2D gastrulation embry- colony outer edge, ectodermal cells in the colony centre, and meso- oid with mouse EpiLCs92. Importantly, findings from this mouse dermal and endodermal cells forming two layers in between18,85,86. embryoid have been compared to the mouse embryo, providing a This multicellular pattern is consistent as in the gastrulating mam- more direct validation, which for obvious reasons is not possible malian embryo. However, the fate territories in the two-dimensional for human gastrulation embryoids. Micropatterning can be read- (2D) gastrulation model are adjacent on a 2D surface rather than lay- ily combined with other bioengineering approaches. For example, ered one on top of the other as in in vivo. Although the 2D geometry recent work has shown that overlaying gradients of exogenous is artificial and distinct from the three-dimensional (3D) topology ligands on micropatterned 2D gastrulation embryoids can bias the of mammalian embryos, the reproducibility and compatibility with resulting fate pattern in a reproducible way93. Specifically, a gradi-

138 Nature Materials | VOL 20 | February 2021 | 132–144 | www.nature.com/naturematerials NaTuRe MaTeRials Review Article ent of BMP4 (and in some cases a counteracting gradient of BMP self-organization96. D–V patterned neural sacs mimicking human antagonist NOGGIN) generated in a microfluidic device induced NT development were recently demonstrated using primed hPSCs axially organized patterning of the germ layers along the gradient, under a culture condition similar to that used for mESCs29. breaking the radial symmetry of 2D circular colonies93. Recently, several 2D models have been reported to recapitulate patterning of a considerable portion of the ectodermal germ layer. 3D models of gastrulation. In addition to blastoids, mESCs and One study with primed hPSCs showed that regional patterning mTSCs can be cultured together in conditions that promote their of neural crest and NE cells could be created on micropatterned self-organization to model post-implantation mouse development. surfaces, mimicking neural induction as the first step of the neu- In such models, rather than forming structures that morphologically rulation process, and that this emergent regional patterning was resemble the mouse blastocyst, mESCs and mTSCs form separate regulated by mechanical control of BMP-SMAD signalling24. Other compartments before fusing together. Each compartment under- recent studies have recapitulated patterning of all four major fates goes lumenogenesis, with the resulting lumens merging together, within the ectoderm during neural induction—neural, neural crest, resulting in a structure resembling the egg cylinder stage mouse placode and epidermis25,97. Modulating both BMP and WNT sig- embryo (referred to as the ETS embryoid)19. Remarkably, the ETS nalling enabled control over the fates that emerged at the border embryoid initiates developmental events mimicking both germ cell between neural and non-neural ectoderm97, and one of these mod- formation and PS development in an asymmetric manner19. This els was shown to be useful for modelling developmental effects is surprising as VE, which is critical for A–P symmetry breaking of the mutations that cause Huntington’s disease25. In the future, in vivo, is not present in the ETS embryoid, although further studies models that recapitulate not only regional fate patterning, but also show that adding XEN cells into the ETS embryoid improves this morphogenesis involved in neural plate folding and neural fold clo- model (called the ETX embryoid)23. sure, could both lead to new fundamental knowledge of this stage of A 3D embryoid for modelling symmetry breaking of the EPI has human development and provide essential systems for research into also been developed starting from primed hPSCs26. In this model, human NT closure defects. hPSCs grown in 3D and treated with a low dose of BMP4 form lumenal sacs before polarizing into two opposing regions displaying Bioengineering tools to control embryoid development gene expression patterns associated with ectoderm and mesoderm Mammalian embryogenesis is a context-dependent process, involv- induction26. This observation is similar to cell fate patterning that ing interactions between multiple, co-emerging embryonic and emerges from the 2D human gastrulation embryoid18. However, as extraembryonic cell lineages that are intricately organized in three the initial degree of symmetry is higher in 3D (sphere versus disc), dimensions. This 3D context provides spatial boundary conditions, the development of the 3D human gastrulation embryoid involves as well as biochemical and biomechanical inputs and positional spontaneous symmetry breaking while in 2D it does not. information that are often absent in conventional 2D culture ves- Finally, an embryoid model beginning from only mESCs sels. Although mouse and human stem cells can be efficiently differ- or primed hPSCs has been shown to be able to model the entiated into specialized cell types under classical 2D cultures, poor post-gastrulation development of the posterior portion of the control over initial seeding conditions and tissue growth lead to dis- mouse and human embryos, respectively17,21,28,94. Growing these organized cell fate patterns and tissue shapes. Bioengineering tools cells in aggregates of defined size and exposing them to a prop- such as microfluidics or microfabricated cell culture substrates have erly timed pulse of WNT activation leads to the formation of a been proven highly effective to ‘reconstruct’, in a bottom-up fash- tail-bud-like structure on one end of the aggregate that continues ion, the missing 3D physical and biochemical context of the early with axial organization, somitogenesis, PGC specification and embryo (Fig. 4). Indeed, recent advances in bioengineering and even NT formation17,21,28,94. These structures, known as gastruloids, biomaterials not only promote the reproducibility of embryoids, recapitulate some essential features of A–P axial patterning of the for example, to facilitate the development of quantitative assays, but post-gastrulation mouse and human embryos as revealed by HOX also enable systematic studies of how the complex array of extrinsic gene expression and somite formation21,28,94. The mouse gastruloid inputs influences embryonic development. can even be coaxed to generate a primitive beating heart following pathways similar to those of the mouse embryo95. These mouse and Micropatterning to control embryoid shape. The simplest human gastruloids all lack anterior structures, such as the forebrain, and perhaps most adopted approach to influence multicellular likely to be due to the posteriorizing effect of WNT signalling. self-organization is based on cultures of cell colonies selectively on While the 3D embryoid models of gastrulation show remark- cell adhesive substrates that are designed to spatially control tissue able emergence of patterning and morphogenesis, they lack the size and shape. This can be readily achieved through micropattern- controllability and reproducibility of the 2D gastrulation models. ing, a classical microfabrication technique widely used to study how Bioengineering approaches, which can control cell–cell interactions cell or tissue shape affects cellular phenotypes98. As illustrated in 2D and modulate symmetry breaking and patterning, as in the micro- embryoid models, 2D micropatterning provides important advan- fluidic PASE27, will be useful for improving the controllability and tages as an assay technology, given their scalability and reproduc- reproducibility of the 3D gastrulation models. ibility, coupled with the ability to manipulate culture conditions and the simplicity of live imaging. Importantly, 2D micropattern- Models of neurulation. The nervous system acquires its form ing can be integrated with microfluidics93 and cell mechanics tools and pattern during the neurulation stage. Several stem-cell-based (such as micropost force sensors or traction force microscopy99) for neurulation models have been developed, which focus on the ecto- dynamic, quantitative measurements and perturbations of soluble dermal germ layer, the source of the nervous system. One of the biochemical signals and insoluble biophysical cues. These unique earliest studies showed that 3D spherical lumenal sacs composed of features have not been fully exploited to date, but are important for neuroepithelial (NE) cells, reminiscent of the NT, could be grown future studies to examine the roles of tissue geometry and mechani- from single mESCs under appropriate neural differentiation condi- cal forces in influencing cell signalling and cell–cell communication tions15. These neural sacs were entirely dorsal in character but could to regulate patterning in 2D embryoids. be patterned by exogenous ventralizing or posteriorizing signals. Using similar microfabrication approaches, micropatterning can Optimizing the gel matrix in which neural sacs were embedded also be extended from 2D to 3D, such as to embed cells within microscale improved their dorsal–ventral (D–V) patterning, showing the power cavities in soft materials such as hydrogels or viscoelastic polymers. of bioengineering approaches to optimize conditions for embryoid This approach has been demonstrated for tubular mammary epithe-

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a b c d

Fig. 4 | Bioengineering tools to promote multicellular interaction and self-organization in embryoid development. a, Micropatterning to generate 2D circular colonies of hPSCs to model germ layer patterning during gastrulation. Immunofluorescence image shows emergence of concentric gene expression regions, mimicking development of the germ layers (SOX2+ ectoderm, blue; TBXT+ mesendoderm, red) as well as a GATA3+ extraembryonic layer (green). b, Microwell array to promote cell aggregation and development of mouse blastoids. Top: a microwell array composed of agarose hydrogel to promote aggression of mESCs and mTSCs. Bottom: merged image showing two blastoids, with a layer of mTSCs surrounding a cavity and a cluster of mESCs mimicking the inner cell mass. Immunostaining: NANOG (red) and GATA6 (green). c, Microfluidics to control spatiotemporal morphogen signalling and tissue patterning. Bright-field (top) and immunofluorescence (bottom) images of an array of post-implantation amniotic sac embryoids (PASEs), showing molecular asymmetry and tissue patterning, with TFAP2A+ amniotic cells on one pole (green) and TBXT+ gastrulating cells (magenta) on the opposite pole. d, Chemically and physically defined hydrogels for 3D embryoid development. Immunofluorescence image of a 3D human gastrulation embryoid for modeling epiblast morphogenesis and patterning (SOX2, green; TBXT, magenta). Credit: A. Yoney/E. D. Siggia (a); N. Rivron (b); Y. Zheng (c); M. Simunovic (d)

lia, shedding light on mechanisms of branching morphogenesis100. embryoid development. Existing data suggest its effect on cell When single primed hPSCs are grown in microcavities overlaid polarity, paracrine signalling, the actin cytoskeleton, and mecha- with Matrigel, the cells self-organize and form a single central lumen notransduction, which are known to regulate classic developmental with a defined geometry83. Such tissues might serve as precursors signalling events. for the generation of new embryoids, for example, for modelling NT patterning along the A–P and/or D–V axes. Microfluidics for establishing signalling centres and gradients and stem cell niches. Whereas micropatterned substrates and Microwell arrays for controlling initial cell aggregation. Another microwell arrays offer an effective means to control cell colony simple yet useful microfabrication approach for improving the shape and aggregate composition, the environment in which the consistency of embryoid development is the microwell array. The cells are grown is typically isotropic and static, and thus poorly microwell array can be used to trap cells in suspension to pro- suited to recapitulate the spatiotemporal dynamics of morpho- mote their initial aggregation into spheroids of controlled sizes gen signalling operating in vivo. Because of its ability to precisely and multicellular compositions. This approach, for example, has manipulate tiny quantities of fluids and establish dynamic chemi- been exploited to reproducibly generate blastoids from mESC/ cal gradients, microfluidics offers exciting opportunities to con- mTSC aggregates in microwell arrays composed of agarose hydro- trol morphogen signalling in space and time such as to establish gels fabricated by micromolding with poly-dimethylsiloxane artificial signalling centres to direct multicellular self-organization (PDMS) stamps22. Along similar lines, the AggreWell plate, a com- and patterning. mercial microwell array, was used to improve the reproducibility An example along this line reported the development of micro- and efficiency in generating ETX embryoids and mEPSC-based fluidic devices to expose micropatterned 2D gastrulation embryoids blastoids23,30,82. to linear morphogen gradients generated via passive diffusion (that For both micropatterning and microwell arrays, their impact is, a ‘source and sink’ type gradient system)93. Beyond establishing on embryoid development seems to derive from their influence on controlled biomolecular gradients, microfluidics offers a power- the determination of the initial cell number in each colony and the ful way to optimize and standardize advanced embryoid cultures, geometric boundaries of the colony. Colony geometry can directly as demonstrated by the microfluidic PASE27. This and similar influence cell signalling and cell–cell communication through microengineered 3D culture systems should be particularly useful regulatory mechanisms involving dynamic morphogenetic cues for designing multicellular embryoid systems with the robustness and diffusible signals24,86,87. It remains elusive how the initial num- and scalability needed in translational applications such as high- ber of cells (or cell density) in each cell colony affects progressive content screening.

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It is worth noting that artificial signalling centres in multicellu- models generated from mESCs15,96. By systematically screening PEG lar self-organization and development can also be established using matrices of variable stiffness, degradability and ECM composition, microbeads loaded with signalling molecules101, optogenetics102, the authors identified a parameter window in which apico-basally or through co-culture with morphogen-secreting cells103. A recent polarized NE sacs with proper D–V patterning robustly emerged, work demonstrated optogenetic stimulation for local activation of with an efficiency greater than achieved in Matrigel96. More recently, WNT signals in both 2D and 3D human gastrulation models to synthetic hydrogels were applied to the 3D human gastrulation drive mesendoderm differentiation102. In another work, an induc- embryoid, by using two commercially available hydrogel systems ible Shh-producing cell aggregate was embedded at one pole of an (physically crosslinked PNIPAAm-PEG gel and an Fmoc-based hPSC spheroid, mimicking a developmental organizer, to promote supramolecular gel) that were admixed with Matrigel26. Beyond ordered self-organization along D–V and A–P axes in a forebrain assisting translational applications of embryoids, the exquisite mod- organoid model103. ularity of such chemically and physically defined hydrogel systems will facilitate a systematic dissection of the independent roles of Advanced biomaterials to mimic the extracellular matrix. extrinsic ECM factors (including matrix stiffness, porosity, degrad- Mammalian development involves not only cell–cell interactions, ability and ECM composition) in early development. but also cell–ECM interactions that guide embryonic organization, cellular differentiation and morphogenesis. The ECM is synthesized Conclusions and future directions and secreted by embryonic cells beginning at the earliest stages of Understanding human development has been one of the central development. Providing adhesive substrates in a 3D context, the goals of modern biology. To circumvent the limited availability of ECM further defines tissue boundaries to guide cell migration and human samples, conventional mammalian developmental biol- functions as a dynamic repository for growth factors to regulate mor- ogy studies have relied heavily on animal models, including NHP phogen signalling. Importantly, embryonic cells sense and respond monkeys. In all of these models, the need for in utero development to the 3D organization and physical properties of the ECM through prevents precise manipulations and high-resolution observation. As mechanotransductive processes involving integrin-mediated adhe- a matter of fact, there will never be sufficient embryonic materi- sions and the intracellular actin cytoskeleton104. An exciting con- als—from humans, NHP monkeys, or other mammalian species— temporary research question is how these mechanotransduction available for quantitative assays with a level of resolution offered processes interact with growth-factor-mediated developmental sig- by synthetic in vitro embryoid systems. As a bottom-up approach nalling to regulate cellular differentiation and patterning104. using stem cells to model embryonic development without using The importance of basement membrane-mediated integrin sig- intact embryos, embryoids are quickly becoming an essential exper- nalling in transforming amorphous EPI cells into an apico-basally imental tool for advancing mammalian embryology. In particular, polarized lumenal EPI sac was first shown in the peri-implantation human embryoids are the only method available to study human mouse embryo105. Indeed, the use of 3D ECM cultures to promote embryological events between the onset of gastrulation and 4–5 the development of mESCs and primed hPSCs into lumenal EPI-like weeks post-fertilization when the earliest fetal tissues from elective structures has been an important first step for the development of terminations are available. By this time point, the primordia of most different embryoids19,20,23,26. In view of these data, how can one then of the major organ systems have formed in the recognizable fetal explain the remarkable level of self-organization and patterning seen body, so it is imperative to develop alternative models to understand in the mouse and human gastruloids grown in suspension, that is, the origins of the human body plan. without a surrounding 3D matrix support13,17,21? Two recent papers The development of embryoids integrates knowledge and meth- that report the exposure of mouse gastruloids to low percentage odologies from stem cell biology, developmental biology, synthetic Matrigel at a later culture time point might shed light on the role of biology, tissue engineering and bioengineering. Coupled with the ECM in morphogenesis in the gastruloid28,94. Intriguingly, instead ease of genetically modifying stem cell lines, the ability to manipu- of observing gene expression patterns in the absence of any visible late culture conditions and the simplicity of live imaging, embry- morphogenesis, as in mouse gastruloids derived in suspension cul- oids are becoming robust and attractive systems to disentangle ture17,21, the provision of Matrigel resulted in the development of cellular behaviours and signalling interactions that drive mam- somites and a NT28,94, suggesting that fate patterns could arise even malian embryogenesis. Using lineage and signalling reporter lines, in morphologically rather disorganized tissues and elaborate mor- embryoids offer exciting experimental systems to study pattern phogenesis and tissue formation might be dependent on physical formation, morphogenesis, cell differentiation, and growth, and contacts with ECM. However, whether Matrigel exerts its function how these developmental processes are dynamically regulated and in the mouse gastruloid through adhesive signalling or mechanical coordinated during embryonic development. Embryoids are also interactions or both remains to be elucidated. useful for elucidating intracellular signalling dynamics and gene All of the above examples have relied on native ECM isolated regulatory networks and their cross-regulation with cell mechanics from animal tissues, in particular Matrigel and Geltrex, basement and morphogenetic signals during embryonic development and for membrane extracts derived from mouse tumour tissues. The main studying classic developmental biology questions, such as symme- limitations of these native ECM, for example, its batch-to-batch try breaking, scaling and induction. variability and potential immunogenicity, are widely documented. Directed differentiations of hPSCs towards clinically relevant cell However, despite sizeable efforts in biomaterial development over lineages using conventional 2D cultures have made remarkable prog- the past decade, it has not yet been possible to identify synthetic ress over the last two decades. Directed differentiations of hPSCs are alternatives that can completely replace native ECM in 3D cultures. largely based on developmental biology knowledge generated from However, some progress has been reported in the use of Matrigel model organisms to optimize growth and differentiation factors to alternatives for embryoid cultures. For example, scientists utilized modulate relevant developmental signalling pathways. However, fibrin matrices, a clinically approved biomaterial generated from intricate cell–cell interactions involved in embryonic development, fibrinogen, to coax mESC colonies to differentiate and form spa- which are important for lineage specification and functional mat- tially organized germ layers16. Interestingly, the authors reported the uration, are often missing and difficult to recapitulate in conven- roles of matrix dimensionality, stiffness, as well as cell–cell adhesion tional 2D cultures. Thus, it is possible that continuous development in promoting germ layer self-organization16. of human embryoids can lead to advanced 3D cultures in which An approach based on chemically defined, poly(ethylene gly- human stem cells can undergo successive developmental stages to col) (PEG)-based hydrogels was explored in the context of 3D NT produce tissue progenitors and fully differentiated cells with better

Nature Materials | VOL 20 | February 2021 | 132–144 | www.nature.com/naturematerials 141 Review Article NaTuRe MaTeRials fidelity to their in vivo counterparts in terms of gene expression, ing stem cell pluripotency. ‘Organism-level’, high-throughput, epigenetics and function. embryoid-based screening pipelines are likely to emerge in the Nonetheless, the widespread utility of embryoids and their near future. We also envision that an important next technological broad impact on human embryology and reproductive medicine milestone is to achieve a human embryoid that can recapitulate the will depend on continuous improvements of their controllability, entire gastrulation process and the development of the trilaminar scalability, reproducibility and standardization and the commercial germ disc containing the three organized definitive germ layers. availability of culture platforms used for embryoid development. Validation of findings from embryoids using in vivo controls More sophisticated embryoid platforms, such as the microfluidic will be important to evaluate their developmental relevance92. PASE, will require additional collaborative efforts between bioengi- However, this is challenging for human embryoids that aim to reca- neers and stem cell and developmental biologists for their dissemi- pitulate post-gastrulation human development, given the scarcity nation to the broad research community. In principle, embryoids of relevant human embryo data108. This challenge will be partially can be integrated with multi-well plate formats to achieve highly addressed by the recent progress of NHP monkey embryo stud- parallelized assays compatible with existing automation workflows ies10–12, which provide quantitative transcriptomic and epigenomic and screening infrastructure. profiles of monkey cells at post-gastrulation developmental stages. Mouse embryoids, which contain all embryonic and extraem- Nonetheless, it becomes imperative to establish a molecular and bryonic lineages and their correct organizations to mimic mouse cellular standard to assess the authenticity and equivalency and development from pre-implantation to early gastrulation, have establish developmental identities of human embryoids compared been successfully developed19,22,23. Since culture protocols are avail- to their in vivo counterparts. This might require the current 14-day able to enable whole mouse embryos to develop in vitro from the rule governing the in vitro human embryo research to be extended pre-gastrula to the early organogenesis stages106, it is conceivable to a post-gastrulation developmental stage9. that mouse embryoids will eventually be able to mimic whole Accompanying the emergence of different human embryoids, mouse embryonic development to the organogenesis stages. there are ongoing discussions and recommendations from the Compared to mouse embryoids, human embryoids developed bioethics community on their regulation109,110. Even though the so far have only used primed hPSCs to model post-implantation existing human embryoids are far from being equivalent to human developmental events associated with the EPI lineage. It is foresee- embryos, as we continue to improve and generate new human able that as hTSCs and human hypoSC lines become available and embryoids, they are expected to more closely mimic human further authenticated, these extraembryonic cells will be integrated embryos, in terms of cell organization, morphogenesis and gene into existing human embryoids (as in the mouse blastoids and ETS expression. The continuous development and progression of this and ETX embryoids19,22,23), allowing their prolonged and organized nascent field will inevitably lead to important bioethical ques- development and studies of the roles of embryonic–extraembryonic tions: what should the ethical status of human embryoids be and interactions in guiding implantation, placentation, embryonic pat- how should they be regulated? What does determine the devel- terning and gastrulation. Another future direction will be to leverage opmental potential of human embryoids in culture and equally hPSCs possessing developmental potency for both embryonic and as important, can it be functionally assessed? Discussions about extraembryonic cell lineages (such as naive hPSCs and hEPSCs) and these questions are clearly out of the scope of this Review, and identify suitable culture conditions to guide their development into the readers are referred to recent commentaries elsewhere109,110. embryoids that contain organized embryonic and extraembryonic Currently there is little explicit regulation of human embryoid structures. Architecturally and functionally competent endometrial research. However, a consensus among researchers working in culture systems are available using both human primary cultures this field (including the authors) has urged regulators to prohibit and established cell lines107. In the future, it will be important to implantation of human embryoids into mammalian uterus and use human embryoids containing TE-like cells coupled with endo- ban the use of human embryoids for reproductive purposes109,110. metrial culture systems to model human implantation and placen- As this nascent field moves forward, we should keep in mind tation, in the hope of understanding the interrelationship between social responsibility as an essential part of the responsible con- embryonic and placental development. duct of research. Transparency and effective engagement with all We envision that continuous developments of mouse and human stakeholders including the public is essential to ensure that prom- embryoids will incorporate new advances of developmental biol- ising avenues for research proceed with due caution, especially ogy, stem cell biology and bioengineering and will lead to improved given the complexity and rapid progress of this field. understanding of intracellular signalling and cell fate dynamics at the single-cell resolution, extracellular movement of developmental Received: 14 April 2020; Accepted: 14 September 2020; signals at cellular and tissue scales, and embryonic–extraembryonic Published online: 16 November 2020 interactions and their critical roles in guiding embryonic develop- ment. It is foreseeable that advanced approaches integrating differ- References ent bioengineering tools will further promote the controllability 1. Solnica-Krezel, L. (ed.) 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M.P.L.’s work is supported by the École org/10.1101/2020.03.04.974949 (2020). Polytechnique Fédérale de Lausanne, the National Center of Competence in Research 95. Rossi, G. et al. Embryonic organoids recapitulate early heart organogenesis. (NCCR) ‘Bio-Inspired Materials’, and the Swiss National Science Foundation Sinergia Preprint at https://doi.org/10.1101/802181 (2019). Grant (no. 3189956). The authors apologize to colleagues whose work they could not cite 96. Ranga, A. et al. Neural tube morphogenesis in synthetic 3D owing to space restrictions. microenvironments. Proc. Natl Acad. Sci. USA 113, E6831–E6839 (2016). 97. Britton, G. et al. A novel self-organizing embryonic stem cell system reveals signaling logic underlying the patterning of human ectoderm. Development Author contributions 146, dev179093 (2019). J.F., A.W. and M.P.L. wrote the manuscript. All authors edited and approved the 98. McBeath, R. et al. Cell shape, cytoskeletal tension, and RhoA regulate stem manuscript. cell lineage commitment. Dev. Cell 6, 483–495 (2004). 99. Lam, R. H. W., Sun, Y., Chen, W. & Fu, J. Elastomeric microposts integrated Competing interests into microfuidics for fow-mediated endothelial mechanotransduction The authors declare no competing interests. analysis. Lab Chip 12, 1865–1873 (2012). 100. Nelson, C. M. et al. Tissue geometry determines sites of mammary branching morphogenesis in organotypic cultures. Science 314, Additional information 298–300 (2006). Correspondence should be addressed to M.P.L., A.W. or J.F. 101. Habib, S. J. et al. A localized Wnt signal orients asymmetric stem cell Reprints and permissions information is available at www.nature.com/reprints. division in vitro. Science 339, 1445–1448 (2013). 102. Repina, N. A. et al. Optogenetic control of Wnt signaling for modeling early Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in embryogenic patterning with human pluripotent stem cells. Preprint at published maps and institutional affiliations. https://doi.org/10.1101/665695 (2019). © Springer Nature Limited 2020

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