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Many mammals can control the timing of gestation and birth by pausing embryonic development at the blastocyst stage. It is unknown whether the capacity to pause development is conserved, in general across mammals, and more specifically in humans. Activity of the growth regulating mTOR pathway governs developmental pausing in the mouse. Here we show a stage-specific capacity to delay the progression of human development via mTOR inhibition. In this context, human blastoids and pluripotent stem cells in naive and naive-like, but not primed, states can be induced to enter a dormant state, which is reversible at the functional and molecular level. Comparative analysis of mouse and human longitudinal response to mTORi revealed distinct temporal dynamics and metabolic requirements of dormancy in each species. Mouse and human blastocysts show similar tissue-specific patterns of mTOR pathway activity, suggesting that the mTOR pathway may be a conserved regulator of blastocyst development and timing in both species. Our results raise the possibility that the developmental timing of the human embryo may be controllable, with implications for reproductive therapies.

Embryogenesis is supported by dynamic loops of cellular interactions. Here, we create a partial mouse embryo model to elucidate the principles of epiblast (Epi) and extra-embryonic endoderm co-development (XEn). We trigger naive mouse embryonic stem cells to form a blastocyst-stage niche of Epi-like cells and XEn-like cells (3D, hydrogel free and serum free). Once established, these two lineages autonomously progress in minimal medium to form an inner pro-amniotic-like cavity surrounded by polarized Epi-like cells covered with visceral endoderm (VE)-like cells. The progression occurs through reciprocal inductions by which the Epi supports the primitive endoderm (PrE) to produce a basal lamina that subsequently regulates Epi polarization and/or cavitation, which, in return, channels the transcriptomic progression to VE. This VE then contributes to Epi bifurcation into anterior- and posterior-like states. Similarly, boosting the formation of PrE-like cells within blastoids supports developmental progression. We argue that self-organization can arise from lineage bifurcation followed by a pendulum of induction that propagates over time.

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.

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals. Neagu, van Genderen and Escudero et al. show that simultaneous inhibition of WNT and MEK signalling maintains a naive-primed intermediate pluripotency state in vitro, which displays features of the mouse embryonic rosette.

The embryo instructs the allocation of cell states to spatially regulate functions. In the blastocyst, patterning of trophoblast (TR) cells ensures successful implantation and placental development. Here, we defined an optimal set of molecules secreted by the epiblast (inducers) that captures in vitro stable, highly self-renewing mouse trophectoderm stem cells (TESCs) resembling the blastocyst stage. When exposed to suboptimal inducers, these stem cells fluctuate to form interconvertible subpopulations with reduced self-renewal and facilitated differentiation, resembling peri-implantation cells, known as TR stem cells (TSCs). TESCs have enhanced capacity to form blastoids that implant more efficiently in utero due to inducers maintaining not only local TR proliferation and self-renewal, but also WNT6/7B secretion that stimulates uterine decidualization. Overall, the epiblast maintains sustained growth and decidualization potential of abutting TR cells, while, as known, distancing imposed by the blastocyst cavity differentiates TR cells for uterus adhesion, thus patterning the essential functions of implantation.

Formulating sound and acceptable embryo research policy remains challenging especially in a pluralistic world. This challenge has acquired a new dimension of complexity with the advent of so-called embryo models, which are derived from stem cells. In this article, we present a normative strategy to facilitate the process of sound policy-making in the field of human embryology. This strategy involves seeking neutral agreements on higher level theories and doctrines as well as seeking agreements on the level of concrete policy proposals. We call this strategy: going high and low. By going high and low, the plurality of reasonable moral and epistemic convictions of stakeholders involved in the domain of human embryology is respected while the process of policy-making in this area is improved.

In our 20th anniversary year, we reflect on how fields have changed since our first issue and here look to the future. In this collection of Voices, our writers speculate on the future: in terms of philosophy, cell states, cell processes, and then how to model cell systems.

SUMMARY Lineage reconstruction is central to understanding tissue development and maintenance. To overcome the limitations of current techniques that typically reconstruct clonal trees using genetically encoded reporters, we report scPECLR, a probabilistic algorithm to endogenously infer lineage trees at a single-cell-division resolution by using 5-hydroxymethylcytosine (5hmC). When applied to 8-cell pre-implantation mouse embryos, scPECLR predicts the full lineage tree with greater than 95% accuracy. In addition, we developed scH&G-seq to sequence both 5hmC and genomic DNA from the same cell. Given that genomic DNA sequencing yields information on both copy number variations and single-nucleotide polymorphisms, when combined with scPECLR it enables more accurate lineage reconstruction of larger trees. Finally, we show that scPECLR can also be used to map chromosome strand segregation patterns during cell division, thereby providing a strategy to test the “immortal strand” hypothesis. Thus, scPECLR provides a generalized method to endogenously reconstruct lineage trees at an individual-cell-division resolution., In brief Wangsanuwat et al. develop a probabilistic algorithm to reconstruct cellular lineages at an individual-cell-division resolution by using strand-specific 5hmC measurements in single cells, and combine it with the development of a single-cell multi-omics technology to quantify 5hmC and genomic DNA from the same cell to reconstruct larger lineage trees., Graphical Abstract

Ultrasound-triggered drug-loaded microbubbles have great potential for drug delivery due to their ability to locally release drugs and simultaneously enhance their delivery into the target tissue. We have recently shown that upon applying ultrasound, nanoparticle-loaded microbubbles can deposit nanoparticles onto cells grown in 2D monolayers, through a process that we termed "sonoprinting". However, the rigid surfaces on which cell monolayers are typically growing might be a source of acoustic reflections and aspherical microbubble oscillations, which can influence microbubble-cell interactions. In the present study, we aim to reveal whether sonoprinting can also occur in more complex and physiologically relevant tissues, by using free-floating 3D tumor spheroids as a tissue model. We show that both monospheroids (consisting of tumor cells alone) and cospheroids (consisting of tumor cells and fibroblasts, which produce an extracellular matrix) can be sonoprinted. Using doxorubicin-liposome-loaded microbubbles, we show that sonoprinting allows to deposit large amounts of doxorubicin-containing liposomes to the outer cell layers of the spheroids, followed by doxorubicin release into the deeper layers of the spheroids, resulting in a significant reduction in cell viability. Sonoprinting may become an attractive approach to deposit drug patches at the surface of tissues, thereby promoting the delivery of drugs into target tissues. ispartof: Journal Of Controlled Release vol:316 pages:79-92 ispartof: location:Netherlands status: published

Summary The ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process., In this perspective, Rossant, Clark, and colleagues outline the process and deliberations that led to the updated ISSCR Guidelines for stem cell research involving the 14-Day rule, stem cell-based embryo models, and in vitro gametogenesis.

Summary The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here., The updated Guidelines for Stem Cell Research and Clinical Translation describe the ethical principles and best practices for basic, translational, and clinical research involving stem cells and human embryos. The updated Guidelines include new recommendations to address the recent scientific advances involving embryos, stem cell-based embryo models, chimeras, organoids, and genome editing.

SUMMARYHuman trophoblast stem cells (hTSC) derived from blastocysts and first-trimester cytotrophoblasts offer an unprecedented opportunity to study the human placenta. However, access to human embryos and first trimester placentas is limited thus preventing the establishment of hTSC from a variety of genetic backgrounds associated with placental disorders. In the present study, we show that hTSC can be generated from numerous genetic backgrounds using post-natal cells via two alternative methods: (I) somatic cell reprogramming of adult fibroblasts using the Yamanaka factors, and (II) cell fate conversion of naive and extended pluripotent stem cells. The resulted induced and converted hTSC (hiTSC/hcTSC) recapitulated hallmarks of hTSC including long-term self-renewal, expression of specific transcription factors, transcriptome-side signature, and the potential to differentiate into syncytiotrophoblast and extravillous trophoblast cells. We also clarified the developmental stage of hTSC and show that these cells resemble post-implantation NR2F2+ cytotrophoblasts (day 8-10). Altogether, hTSC lines of diverse genetics origins open the possibility to model both placental development and diseases in a dish.HighlightsReprogramming of human somatic cells to induced hTSC with OSKMConversion of naive and extended hPSC to hTSCGenetic diversity of hTSC linesDevelopmental matching of hTSC in the peri-implantation human embryo

Lineage reconstruction is central to understanding tissue development and maintenance. While powerful tools to infer cellular relationships have been developed, these methods typically have a clonal resolution that prevent the reconstruction of lineage trees at an individual cell division resolution. Moreover, these methods require a transgene, which poses a significant barrier in the study of human tissues. To overcome these limitations, we report scPECLR, a probabilistic algorithm to endogenously infer lineage trees at a single cell-division resolution using 5-hydroxymethylcytosine. When applied to 8-cell preimplantation mouse embryos, scPECLR predicts the full lineage tree with greater than 95% accuracy. Further, scPECLR can accurately extract lineage information for a majority of cells when reconstructing larger trees. Finally, we show that scPECLR can also be used to map chromosome strand segregation patterns during cell division, thereby providing a strategy to test the “immortal strand” hypothesis in stem cell biology. Thus, scPECLR provides a generalized method to endogenously reconstruct lineage trees at an individual cell-division resolution.

During germ cell development, cells undergo a drastic switch from mitosis to meiosis to form haploid germ cells. Sequencing and computational technologies now allow studying development at the single-cell level. Here we developed a multiplexed trajectory reconstruction to create a high-resolution developmental map of spermatogonia and prophase-I spermatocytes from testes of a Dazl-GFP reporter mouse. We identified three main transitions in the meiotic prophase-I: meiotic entry, the meiotic sex chromosome inactivation (MSCI), and concomitant pachytene activation. We validated the key features of these transitionsin vivousing single molecule FISH. Focusing on MSCI, we found that 34% of sex chromosomal genes are induced shortly before MSCI, that silencing time is diverse and correlates with specific gene functions. These highlight a previously underappreciated level of regulation of MSCI. Finally, we found that spermatozoal genes in pachytene are activated in a temporal pattern reflecting the future anatomic and functional order of the sperm cell. Altogether we highlighted how precise and sequential changes in gene expression regulate cellular states in meiotic prophase-I.

Early mammalian embryos form a blastocyst, a structure comprising embryonic cells surrounded by a trophoblast cyst able to implant into the mother’s uterus. Following subtle symmetry-breaking events (Zhang and Hiiragi, 2018), an axis forms in the blastocyst when the embryonic cells cluster on one inner side of the trophoblast globe and induce the directional proliferation and differentiation of trophoblasts (Gardner, 2000). Trophoblast stem cells (TSCs) are in vitro analogs of early trophoblasts (Tanaka et al., 1998). Here, we show that TSCs contain a range of plastic subpopulations reflecting aspects of trophoblasts states ranging from the blastocyst trophoblasts juxtaposing the embryo (polar trophoblasts) to post-implantation trophoblasts. However, when exposed to a specific combination of embryonic inductive signals, TSCs acquire properties of polar trophoblasts (gene expression, self-renewal) and a more homogeneous, epithelial phenotype. These lines of polar-like TSCs more efficiently form blastoids, which respond to the inner embryonic cells by spontaneously generating an embryonic-abembryonic axis. Altogether, the delineation of requirements and properties of the polar cells of the trophectoderm provides the ground to better recapitulate and dissect embryonic development in vitro.

The early mammalian conceptus (blastocyst) contains two supporting extraembryonic tissues - the trophectoderm and the primitive endoderm (PrE) - that encase and guide the epiblast (Epi) to eventually form the all body. Modifications of the conceptus exposed key genes regulating these tissues co-development. However, the combinations of signalling pathways underlying the interplay of PrE and Epi remains elusive. Stem cell-based models including embryoid bodies and blastoids can be generated in large numbers and subjected to high-content screens. Here, we use combinatorial screens of proteins, GPCR ligands and small molecules to rapidly (72 hours) and efficiently (80%) guide embryoid bodies to form a three-dimensional PrE-/Epiblast-like niche in chemically-defined conditions (gel-free, serum-free). This bipotent niche spontaneously progresses, without growth factors, to form a pro-amniotic cavity surrounded by a polarized Epi covered with parietal and visceral endoderm-like cells. In blastoids, these molecules enhance the ratio and number of Gata6+/Nanog+ cells and promote the survival, expansion and morphogenesis of a post-implantation-like Epiin vitro. Altogether, modelling early development in chemically-defined conditions delineates the pathways sufficient to form a functional PrE/Epiblast niche that fuels post-implantation development.

International discussion must guide research, urge Nicolas Rivron, Martin Pera and colleagues. International discussion must guide research, urge Nicolas Rivron, Martin Pera and colleagues.

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67(iresCreER) allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.

The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that surrounds a fluid-filled cavity sheltering the embryonic cells1. From mouse blastocysts, it is possible to derive both trophoblast2 and embryonic stem-cell lines3, which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal–KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation. Trophoblast and embryonic stem cells interact in vitro to form structures that resemble early blastocysts, and the embryo provides signals that drive early trophectoderm development and implantation.

International audience; Although bone marrow stromal cells (BMSCs) appear promising for cartilage repair, current clinical results are suboptimal and the success of BMSC-based therapies relies on a number of methodological improvements, among which is better understanding and control of their differentiation pathways. We investigated here the role of the cellular environment (paracrine vs juxtacrine signalling) in the chondrogenic differentiation of BMSCs. Bovine BMSCs were encapsulated in alginate beads, as dispersed cells or as small micro-aggregates, to create different paracrine and juxtacrine signalling conditions. BMSCs were then cultured for 21 days with TGFβ3 added for 0, 7 or 21 days. Chondrogenic differentiation was assessed at the gene (type II and X collagens, aggrecan, TGFβ, sp7) and matrix (biochemical assays and histology) levels. The results showed that micro-aggregates had no beneficial effects over dispersed cells: matrix production was similar, whereas chondrogenic marker gene expression was lower for the micro-aggregates, under all TGFβ conditions tested. This weakened chondrogenic differentiation might be explained by a different cytoskeleton organization at day 0 in the micro-aggregates.

3D organoids using stem cells to study development and disease are now widespread. These models are powerful to mimic in vivo situations but are currently associated with high variability and low throughput. For biomedical research, platforms are thus necessary to increase reproducibility and allow high-throughput screens (HTS). Here, we introduce a microwell platform, integrated in standard culture plates, for functional HTS. Using micro-thermoforming, we form round-bottom microwell arrays from optically clear cyclic olefin polymer films, and assemble them with bottom-less 96-well plates. We show that embryonic stem cells aggregate faster and more reproducibly (centricity, circularity) as compared to a state-of-the-art microwell array. We then run a screen of a chemical library to direct differentiation into primitive endoderm (PrE) and, using on-chip high content imaging (HCI), we identify molecules, including regulators of the cAMP pathway, regulating tissue size, morphology and PrE gene activity. We propose that this platform will benefit to the systematic study of organogenesis in vitro.

An accessible and versatile microfabrication platform was demonstrated to build scaffold-free 3D tissues with complex architectures. As a platform for the controlled and uniform formation of multicellular building blocks and tissues, a microwell array system was fabricated using soft lithography methods. Microstructures were copied from a SU-8/silicon wafer via a negative replica of the same in the form of an elastomeric stamp cast from poly(dimethylsiloxane) (PDMS) into an agarose hydrogel The resulting hydrogel chips including the microstructures were transferred into standard 12-well culture plates (12wp) to maximize compatibility with existing laboratory equipment. A single PDMS stamp was able to imprint arrays of several hundreds to thousands of cylindrical microwells per well of a 12wp and with high precision and reproducibility. As a second step, these aggregates were used as multicellular building blocks within cavities of millimeter length-scale hydrogel templates with defined geometry. The formation of stapes-like tissues demonstrates the dimensional flexibility of the bottom-up approach in building larger tissues with precisely defined shape.

Large bone defects naturally regenerate via a highly vascularized tissue which progressively remodels into cartilage and bone. Current approaches in bone tissue engineering are restricted by delayed vascularization and fail to recapitulate this stepwise differentiation toward bone tissue. Here, we use the morphogen Sonic Hedgehog (Shh) to induce the in vitro organization of an endothelial capillary network in an artificial tissue. We show that endogenous Hedgehog activity regulates angiogenic genes and the formation of vascular lumens. Exogenous Shh further induces the in vitro development of the vasculature (vascular lumen formation, size, distribution). Upon implantation, the in vitro development of the vasculature improves the in vivo perfusion of the artificial tissue and is necessary to contribute to, and enhance, the formation of de novo mature bone tissue. Similar to the regenerating callus, the artificial tissue undergoes intramembranous and endochondral ossification and forms a trabecular-like bone organ including bone-marrow-like cavities. These findings open the door for new strategies to treat large bone defects by closely mimicking natural endochondral bone repair.

This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches. Electronic supplementary material The online version of this article (doi:10.1007/s10544-011-9588-5) contains supplementary material, which is available to authorized users.

Tissue engineering aims at replacing or regenerating tissues lost due to diseases or traumas (Langer and Vacanti, 1993). However, mimicking in vitro the physiological complexity of vascularized tissue is a major obstacle, which possibly contributes to impaired healing in vivo. In higher organisms, native features including the vascular network, the lymphatic networks and interstitial flow promote both mass transport and organ development. Attempts to mimic those features in engineered tissues will lead to more clinically relevant cell-based therapies. Aside from current strategies promoting angiogenesis from the host, an alternative concept termed prevascularization is emerging. It aims at creating a biological vasculature inside an engineered tissue prior to implantation. This vasculature can rapidly anastamose with the host and enhances tissue survival and differentiation. Interestingly, growing evidence supports a role of the vasculature in regulating pattern formation and tissue differentiation. Thus, prevascularized tissues also benefit from an intrinsic contribution of their vascular system to their development. From those early attempts are emerging a body of principles and strategies to grow and maintain, in vitro, those self-assembled biological vascular networks. This could lead to the generation of engineered tissues of more physiologically relevant complexity and improved regenerative potential.

A material-based bottom-up approach is proposed towards an assembly of cells and engineered micro-objects at the macroscale. We show how shape, size and wettability of engineered micro-objects play an important role in the behavior of cells on these objects. This approach can, among other applications, be used as a tool to engineer complex 3D tissues of clinically relevant size.

3D cell culture methods confer a high degree of clinical and biological relevance to in vitro models. This is specifically the case with the spheroid culture, where a small aggregate of cells grows free of foreign materials. In spheroid cultures, cells secrete the extracellular matrix (ECM) in which they reside, and they can interact with cells from their original microenvironment. The value of spheroid cultures is increasing quickly due to novel microfabricated platforms amenable to high-throughput screening (HTS) and advances in cell culture. Here, we review new possibilities that combine the strengths of spheroid culture with new microenvironment fabrication methods that allow for the creation of large numbers of highly reproducible, complex tissues.

Physical forces play a major role in the organization of developing tissues. During vascular development, physical forces originating from a fluid phase or from cells pulling on their environment can alter cellular signaling and the behavior of cells. Here, we observe how tissue deformation spatially modulates angiogenic signals and angiogenesis. Using soft lithographic templates, we assemble three-dimensional, geometric tissues. The tissues contract autonomously, change shape stereotypically and form patterns of vascular structures in regions of high deformations. We show that this emergence correlates with the formation of a long-range gradient of Vascular Endothelial Growth Factor (VEGF) in interstitial cells, the local overexpression of the corresponding receptor VEGF receptor 2 (VEGFR-2) and local differences in endothelial cells proliferation. We suggest that tissue contractility and deformation can induce the formation of gradients of angiogenic microenvironments which could contribute to the long-range patterning of the vascular system.

In vitro-generated tissues hold significant promise to mimic healthy and diseased tissues and impact both regenerative medicine and fundamental science. Current technologies allow for the formation of precise multicellular assemblies using, for instance, cell patterning approaches. These approaches lead to the formation of systems that are not necessarily stable and will remodel and reorganize over time, based on physical and/or biological principles (i.e. migration of the cells, shrinkage of the hydrogel). Shapes and patterns are thus not inevitably translated to the final tissue. Besides the technical issues of tissue assembly, there is a need for understanding collective cellular behaviors so as to design tissues prone to predictable and adequate self-deformation, self-remodeling and self-organization. Microfabricated tissues are powerful tools to study, in vitro, these processes. They will help to set the basic principles to rationally design tissues prone to further development and subsequently improve approaches to engineer more complex tissue architectures and functionalities. In this chapter, we will first briefly define and discuss the principles and mechanisms of self-organization during natural tissue development. We will then depict current attempts in studying these principles in in vitro microfabricated tissue models. We will specifically focus on current models using hydrogel templates or supports to assemble cells into primitive patterns. Finally, we will discuss the role of geometries in promoting heterogeneity of biological and physical cues leading to the emergence of self-organized forms.

recapitulating developmental mechanisms in vitro necessitate models of intermediate complexity, between simple 2D culture and complex in vivo models, which integrate both physical and molecular cues. Here, we describe a cheap and simple bottom-up microfabrication method to build 3D millimeter-scale tissues with geometric shapes. These tissues are suitable for long-term culture in tissue-based assays and as implants for clinical applications. In a case study, we recapitulate some mechanisms of vasculogenesis, the assembly of capillary blood vessels.

In vitro-generated tissues hold significant promise in modern biology since they can potentially mimic physiological and pathological tissues. However, these are currently structurally and functionally of limited complexity and necessitate self-organization and recapitulation of tissue development mechanisms in vitro. Tools derived from nano- and microfabrications along with bottom-up strategies are emerging to allow the fabrication of primitive tissues structures that can remodel overtime. Subsequently, clues are accumulating to show that, beyond genetic material, both intrinsic tissue architectures and microenvironmental cues can lead to morphogenesis related mechanisms in vitro. The question arises, however, as how we may design and assemble structures prone to adequate tissue remodeling, predict and manipulate those developmental mechanisms in vitro? Systems integrating architectural, physical and molecular cues will allow more systematic investigation of basic principles of tissue morphogenesis, differentiation or maintenance and will feedback to reproduce the dynamic of tissue development in vitro and form more complex tissues.

Tissue engineering has been an active field of research for several decades now. However, the amount of clinical applications in the field of tissue engineering is still limited. One of the current limitations of tissue engineering is its inability to provide sufficient blood supply in the initial phase after implantation. Insufficient vascularization can lead to improper cell integration or cell death in tissue-engineered constructs. This review will discuss the advantages and limitations of recent strategies aimed at enhancing the vascularization of tissue-engineered constructs. We will illustrate that combining the efforts of different research lines might be necessary to obtain optimal results in the field.

One week after fertilization, human embryos implant into the uterus. This event requires the embryo to form a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form a blastocyst model that we called a blastoid1. Here we show that naive human pluripotent stem cells cultured in PXGL medium2 and triply inhibited for the Hippo, TGF-β and ERK pathways efficiently (with more than 70% efficiency) form blastoids generating blastocyst-stage analogues of the three founding lineages (more than 97% trophectoderm, epiblast and primitive endoderm) according to the sequence and timing of blastocyst development. Blastoids spontaneously form the first axis, and we observe that the epiblast induces the local maturation of the polar trophectoderm, thereby endowing blastoids with the capacity to directionally attach to hormonally stimulated endometrial cells, as during implantation. Thus, we propose that such a human blastoid is a faithful, scalable and ethical model for investigating human implantation and development3,4., Blastoids derived from naive PXGL-cultured human pluripotent stem cells in which Hippo, TGF-β and ERK pathways are inhibited closely recapitulate aspects of blastocyst development, form cells resembling blastocyst-stage cells and thus provide a model system for implantation and development studies.