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Mechanics-guided neuroectoderm patterning is mediated by BMP-SMAD signalling a, Representative immunofluorescence images showing colony central and peripheral zones at day 4 stained for phosphorylated SMAD 1/5 (p-SMAD 1/5). hPS cells were plated at either 20,000 cells cm⁻² (control) or 5,000 cells cm⁻² (low density) and were cultured in neural induction medium supplemented with either DMSO or blebbistatin (Bleb; 10 µM). Red and yellow rectangles highlight selected peripheral and central regions, respectively, where fluorescence intensities of DAPI and p-SMAD 1/5 were measured along white solid lines drawn across these selected areas. Scale bar, 40 µm. Experiments were repeated three times with similar results. b, Percentage of cells with nuclear p-SMAD 1/5 as a function of distance from colony centroid. Based on distance of nuclei from colony centroid, cells were grouped into four concentric zones with equal widths as indicated. The number of colonies analysed was pooled from n = 3 independent experiments. Data were plotted as the mean ± s.e.m. P values were calculated between Bleb versus control and low density versus control using unpaired, two-sided Student’s t-tests. c, Representative immunofluorescence micrographs showing patterned circular single hPS cells with defined spreading areas (500 µm² versus 1,600 µm²) stained for p-SMAD 1/5. White dashed lines mark the cell shape. Scale bar, 20 µm. Experiments were repeated three times with similar results. d, Bar plot showing percentages of cells with dominant nuclear or cytoplasmic p-SMAD 1/5 as a function of cell spreading area. n = 3 independent experiments. Data were plotted as the mean ± s.e.m.
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Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic development. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based ne...
Citations
... [38]. Researchers utilizing a human pluripotent stem cell micropatterning system demonstrated that direct mechanical force on tissue (e.g., stretching) increases cell-adhesion tension, leading to relocalization of β-catenin and activation of Wnt signaling, subsequently regulating mesoderm specification [39] and that patterning of neuroepithelial/neural plate border in a BMP-SMAD dependent manner is also controlled by physical forces (increase in stretching) [40]. Moreover, fluid sheer stress promotes MSC differentiation into osteogenic fate dependent on Ca 2+ and MAPK/ERK signaling [41,42]. ...
Cell fate determination, a vital process in early development and adulthood, has been the focal point of intensive investigation over the past decades. Its importance lies in its critical role in shaping various and diverse cell types during embryonic development and beyond. Exploration of cell fate determination started with molecular and genetic investigations unveiling central signaling pathways and molecular regulatory networks. The molecular studies into cell fate determination yielded an overwhelming amount of information invoking the notion of the complexity of cell fate determination. However, recent advances in the framework of biomechanics have introduced a paradigm shift in our understanding of this intricate process. The physical forces and biochemical interplay, known as mechanotransduction, have been identified as a pivotal drive influencing cell fate decisions. Certainly, the integration of biomechanics into the process of cell fate pushed our understanding of the developmental process and potentially holds promise for therapeutic applications. This integration was achieved by identifying physical forces like hydrostatic pressure, fluid dynamics, tissue stiffness, and topography, among others, and examining their interplay with biochemical signals. This review focuses on recent advances investigating the relationship between physical cues and biochemical signals that control cell fate determination during early embryonic development.
... As a result, cell polarity is established within a single colony. The polarity structure of human pluripotent stem cells (hPSCs) is derived from characteristics similar to epithelial cells, and it exhibits variances in cell shape, secretion of morphogens (Etoc et al., 2016), and the cell cytoskeleton (Xue et al., 2018) depending on the cellular position within the colony (Simunovic and Brivanlou, 2017) (Krtolica et al., 2009). These varying biological responses lead to complex patterning of germ layers in pluripotent stem cells (Bauwens et al., 2008;Warmflash et al., 2014). ...
... Neuruloids derived from both isogenic HD hESCs and homozygote null HTT-/-mutants exhibited highly reproducible phenotypes (Haremaki et al., 2019). In addition, micropattern has been used for neuroectoderm patterning (Xue et al., 2018). Dual SMAD inhibition results in neural induction at the center of micropatterned colony and brief treatment of Chiron induces neural plate border in the periphery. ...
... Neuroectoderm is a transient embryonic tissue that gives rise to the central nervous system, including the brain and spinal cord. Within the neuroectoderm, neural stem cells are formed, which are self-renewing and capable of generating various neural cell types [13]. After the formation of neural stem cells, they begin to self-organize and differentiate further. ...
Brain organoid implications have opened vast avenues in the realm of interdisciplinary research, particularly in the growing field of organoid intelligence (OI). A brain organoid is a three-dimensional (3D), lab-grown structure that mimics certain aspects of the human brain organization and function. The integration of organoid technology with computational methods to enhance the understanding of organoid behavior and to predict their responses to various stimuli is known as OI. The ability of brain organoids to adapt and memorize, is a key area of exploration. OI encapsulates the confluence of breakthroughs in stem cell technology, bioengineering, and artificial intelligence (AI). This chapter delves deep into the myriad potentials of OI, encompassing an enhanced understanding of human cognitive functions, and achieving significant biological computational proficiencies. Such advancements stand to offer a unique complementarity to conventional computing methods. The implications of brain organoids in the OI sphere signify a transformative stride towards a more intricate grasp of the human brain and its multifaceted intricacies. The intersection of biology and machine learning is a rapidly evolving field that is reshaping our understanding of life and health. This convergence is driving advancements in numerous areas, including genomics, drug discovery, personalized medicine, and synthetic biology.
... A different approach to study early human development involves in vitro models that begin with pluripotent stem cells (PSCs) [8][9][10][11][12][13] . For example, 'gastruloids' leverage the self-organizing potential of PSCs 1-4,14 and are distinguished by the formation of derivatives of all three germ layers, as well as by the specification of an anteroposterior axis 3 . ...
Embryonic organoids are emerging as powerful models for studying early mammalian development. For example, stem cell-derived 'gastruloids' form elongating structures containing all three germ layers. However, although elongated, human gastruloids do not morphologically resemble post-implantation embryos. Here we show that a specific, discontinuous regimen of retinoic acid (RA) robustly induces human gastruloids with embryo-like morphological structures, including a neural tube and segmented somites. Single cell RNA-seq (sc-RNA-seq) further reveals that these human 'RA-gastruloids' contain more advanced cell types than conventional gastruloids, including neural crest cells, renal progenitor cells, skeletal muscle cells, and, rarely, neural progenitor cells. We apply a new approach to computationally stage human RA-gastruloids relative to somite-resolved mouse embryos, early human embryos and other gastruloid models, and find that the developmental stage of human RA-gastruloids is comparable to that of E9.5 mouse embryos, although some cell types show greater or lesser progression. We chemically perturb WNT and BMP signaling in human RA-gastruloids and find that these signaling pathways regulate somite patterning and neural tube length, respectively, while genetic perturbation of the transcription factors PAX3 and TBX6 markedly compromises the formation of neural crest and somites/renal cells, respectively. Human RA-gastruloids complement other embryonic organoids in serving as a simple, robust and screenable model for decoding early human embryogenesis.
... Interestingly, channels were observed to be active at both the lumen and the outer edges. These regions are characterized by high mechanical tension, indicated by prominent actin staining and previous work illustrating high traction stress in the outer edges of a micropatterned neuroectoderm model 56 . Our observation highlights the need to consider the site-specific biology of tissues and their unique geometrical and mechanical properties when studying physiological roles of PIEZO1. ...
PIEZO1 channels play a critical role in numerous physiological processes by transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of endogenous PIEZO1 activity and localization in regulating mechanotransduction. To enable physiologically and clinically relevant human-based studies, we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with super-resolution imaging, our chemogenetic approach allows precise visualization of PIEZO1 in various cell types. Further, the PIEZO1-HaloTag hiPSC technology allows non-invasive monitoring of channel activity via Ca ²⁺ -sensitive HaloTag ligands, with temporal resolution approaching that of patch clamp electrophysiology. Using lightsheet imaging of hiPSC-derived neural organoids, we also achieve molecular scale PIEZO1 imaging in three-dimensional tissue samples. Our advances offer a novel platform for studying PIEZO1 mechanotransduction in human cells and tissues, with potential for elucidating disease mechanisms and development of targeted therapeutics.
... In our model, pSmad1/5 levels depend on the local concentration of BMP ligands with constant sensitivity, except for cells at the edge of the colony, which are more sensitive to BMP (SI). This edge effect is similar to that observed in other micropattern protocols 22,28,29 and has been attributed to differential receptor accessibility and mechanical effects. In turn, pSmad1/5 induces the expression of diffusible BMP ligands, based on our observation that endogenous ligand expression is induced upon BMP treatment (Fig. S3C). ...
Developing tissues interpret dynamic changes in morphogen activity to generate cell type diversity. To quantitatively study BMP signalling dynamics in the vertebrate neural tube, we developed a new ES cell differentiation system tailored for growing tissues. Differentiating cells form striking self-organised patterns of dorsal neural tube cell types driven by sequential phases of BMP signalling that are observed both in vitro and in vivo. Data-driven biophysical modelling showed that these dynamics result from coupling fast negative feedback with slow positive regulation of signalling by the specification of an endogenous BMP source. Thus, in contrast to relays that propagate morphogen signalling in space, we uncover a BMP signalling relay that operates in time. This mechanism allows rapid initial concentration-sensitive response that is robustly terminated, thereby regulating balanced sequential cell type generation. Altogether, our study provides an experimental and theoretical framework to understand how signalling dynamics are exploited in developing tissues.
... In addition, graphene has many advantages such as extraordinary biological properties, excellent electrical conductivity, mechanical strength, large surface area, good elasticity, renewability, low cost, and easy acquisition [22][23][24]. In addition to graphene, many derivatives of graphene have been extensively studied due to their similar or complementary properties [25]. The main graphene derivatives include pristine graphene, graphene oxide (GO), reduced graphene oxide (RGO), graphene quantum dots (GQDs), graphene nanosheets, monolayers, multilayer graphene, and graphene-based nanocomposites [26,27]. ...
As bone and joint injuries from various causes become increasingly prominent, how to effectively reconstruct and repair bone defects presents a difficult problem for clinicians and researchers. In recent years, graphene and its derivatives have been the subject of growing body of research and have been found to promote the proliferation and osteogenic differentiation of stem cells. This provides a new idea for solving the clinical problem of bone defects. However, as as numerous articles address various aspects and have not been fully systematized, there is an urgent need to classify and summarize them. In this paper, for the first time, the effects of graphene and its derivatives on stem cells in solution, in 2D and 3D structures and in vivo and their possible mechanisms are reviewed, and the cytotoxic effects of graphene and its derivatives were summarized and analyzed. The toxicity of graphene and its derivatives is further reviewed. In addition, we suggest possible future development directions of graphene and its derivatives in bone tissue engineering applications to provide a reference for further clinical application.
... different from those of tissue patterning, folding, and lumenogenesis seen in previous models [17][18][19][20][21][22][23][24] , remains undetermined. Here we developed a micropatterned gut spheroid generator (μGSG) system for efficient generation of various region-specific gut spheroids from hPSCs. ...
... To explore the mechanism underlying such mechanically enhanced biogenesis of gut spheroids in μGSG, we used μGSG-PFG as a model system henceforth. We first examined whether it is subject to the influence of micropattern size and geometry, which have been found important in guiding embryoid and organoid development [17][18][19][35][36][37][38][39] . Interestingly, spheroid formation efficiency remained unchanged in μGSG featuring circular micropatterns with a broad range of diameters (d = 50-8000 μm), all showing significantly higher efficiency than monolayer-based induction (Fig. 4a, b; Supplementary Fig. 11a). ...
... Spheroid formation efficiency also remains unaffected in μGSG made of rectangular micropatterns with different aspect ratios and areas (Fig. 4c, d). Therefore, μGSG enhances the biogenesis of gut spheroids independent of the size and shape of the micropatterns, implicating a previously unappreciated mechanism unlike the canonical edgesensing mechanism reported in other micropatterned-based models 17,23,35,[39][40][41][42] . ...
Region-specific gut spheroids are precursors for gastrointestinal and pulmonary organoids that hold great promise for fundamental studies and translations. However, efficient production of gut spheroids remains challenging due to a lack of control and mechanistic understanding of gut spheroid morphogenesis. Here, we report an efficient biomaterial system, termed micropatterned gut spheroid generator (μGSG), to generate gut spheroids from human pluripotent stem cells through mechanically enhanced tissue morphogenesis. We show that μGSG enhances the biogenesis of gut spheroids independent of micropattern shape and size; instead, mechanically enforced cell multilayering and crowding is demonstrated as a general, geometry-insensitive mechanism that is necessary and sufficient for promoting spheroid formation. Combining experimental findings and an active-phase-field morphomechanics theory, our study further reveals an instability-driven mechanism and a mechanosensitive phase diagram governing spheroid pearling and fission in μGSG. This work unveils mechanobiological paradigms based on tissue architecture and surface tension for controlling tissue morphogenesis and advancing organoid technology.
... When dissociated single pluripotent stem cells are grown in a pre-patterned geometrically-confined culture surface in a medium supplemented with dual inhibitors for TGF-β and BMP4, concentric zones of progenitors expressing the markers for neural plate and neural plate border are observed. Morphogenetic cues -cell shape and cytoskeletal contractile force -dictate the patterning of the neural plate and neural plate border via BMP-SMAD signaling (Xue et al., 2018). When dissociated single pluripotent stem cells are grown as individual colonies in a medium supplemented with knockout serum replacement, multiple zones of ectodermal cells autonomously form (Hayashi et al., 2016). ...
The telencephalon and eye in mammals are originated from adjacent fields at the anterior neural plate. Morphogenesis of these fields generates telencephalon, optic-stalk, optic-disc, and neuroretina along a spatial axis. How these telencephalic and ocular tissues are specified coordinately to ensure directional retinal ganglion cell (RGC) axon growth is unclear. Here, we report self-formation of human telencephalon-eye organoids comprising concentric zones of telencephalic, optic-stalk, optic-disc, and neuroretinal tissues along the center-periphery axis. Initially-differentiated RGCs grew axons towards and then along a path defined by adjacent PAX2+ VSX2+ optic-disc cells. Single-cell RNA sequencing of these organoids not only confirmed telencephalic and ocular identities but also identified expression signatures of early optic-disc, optic-stalk, and RGCs. These signatures were similar to those in human fetal retinas. Optic-disc cells in these organoids differentially expressed FGF8 and FGF9 ; FGFR inhibitions drastically decreased early RGC differentiation and directional axon growth. Through the RGC-specific cell-surface marker CNTN2 identified here, electrophysiologically excitable RGCs were isolated under a native condition. Our findings provide insight into the coordinated specification of early telencephalic and ocular tissues in humans and establish resources for studying RGC-related diseases such as glaucoma.
... We show that this approach can generate both fast and slow-changing forces, and that rapid short-term actuation reversibly deforms local tissue, while longer-term actuation causes permanent reorganization and tissuescale differential growth. While no preferential direction was previously observed in neural tissue under exogenous actuation 16,36 , our results demonstrate that local actuation guides directional patterning bias. Moreover, while local magnetic actuation is demonstrated in human neural tube organoids here, the use of magnetoids in other multicellular contexts could serve to address a wide variety of mechanobiological questions related to the effects of local forces in development and disease. ...
Tissues take shape through a series of morphogenetic movements guided by local cell-scale mechanical forces. Current in vitro approaches to recapitulate tissue mechanics rely on uncontrolled self-organization or on the imposition of extrinsic and homogenous forces using matrix or instrument-driven stimulation, thereby failing to recapitulate highly localized and spatially varying forces. Here we develop a method for targeted mechanical stimulation of organoids using embedded magnetic nanoparticles. We show that magnetic clusters within organoids can be produced by sequential aggregation of magnetically labeled and non-labeled human pluripotent stem cells. These clusters impose local mechanical forces on the surrounding cells in response to applied magnetic fields. We show that precise, spatially defined actuation provides short-term mechanical tissue perturbations as well as long-term cytoskeleton remodeling in these organoids, which we term "magnetoids". We demonstrate that targeted magnetic nanoparticle-driven actuation guides asymmetric tissue growth and proliferation, leading to enhanced patterning in human neural magnetoids. This approach, enabled by nanoparticle technology, allows for precise and locally controllable mechanical actuation in human neural tube organoids, and could be widely applicable to interrogate the role of local mechanotransduction in developmental and disease model systems.
