Spatial cell fate manipulation of human pluripotent stem cells by controlling the microenvironment using photocurable hydrogel.

Human pluripotent stem cells (hPSCs) dynamically respond to their chemical and physical microenvironment, dictating their behavior. However, conventional in vitro studies predominantly employ plastic culture wares, which offer a simplified representation of the in vivo microenvironment. Emerging evidence underscores the pivotal role of mechanical and topological cues in hPSC differentiation and maintenance. In this study, we cultured hPSCs on hydrogel substrates with spatially controlled stiffness. The use of culture substrates that enable precise manipulation of spatial mechanical properties holds promise for better mimicking in vivo conditions and advancing tissue engineering techniques. We designed a photocurable polyethylene glycol-polyvinyl alcohol (PVA-PEG) hydrogel, allowing the spatial control of surface stiffness and geometry at a micrometer scale. This versatile hydrogel can be functionalized with various extracellular matrix proteins. Laminin 511-functionalized PVA-PEG gel effectively supports the growth and differentiation of hPSCs. Moreover, by spatially modulating the stiffness of the patterned gel, we achieved spatially selective cell differentiation, resulting in the generation of intricate patterned structures.

Collective nuclear behavior shapes bilateral nuclear symmetry for subsequent left-right asymmetric morphogenesis in Drosophila.

Proper organ development often requires nuclei to move to a specific position within the cell. To determine how nuclear positioning affects left-right (LR) development in the Drosophila anterior midgut (AMG), we developed a surface-modeling method to measure and describe nuclear behavior at stages 13-14, captured in three-dimensional time-lapse movies. We describe the distinctive positioning and a novel collective nuclear behavior by which nuclei align LR symmetrically along the anterior-posterior axis in the visceral muscles that overlie the midgut and are responsible for the LR-asymmetric development of this organ. Wnt4 signaling is crucial for the collective behavior and proper positioning of the nuclei, as are myosin II and the LINC complex, without which the nuclei fail to align LR symmetrically. The LR-symmetric positioning of the nuclei is important for the subsequent LR-asymmetric development of the AMG. We propose that the bilaterally symmetrical positioning of these nuclei may be mechanically coupled with subsequent LR-asymmetric morphogenesis.

Emergence of dorsal-ventral polarity in ESC-derived retinal tissue.

We previously demonstrated that mouse embryonic stem cell (mESC)-derived retinal epithelium self-forms an optic cup-like structure. In the developing retina, the dorsal and ventral sides differ in terms of local gene expression and morphological features. This aspect has not yet been shown in vitro Here, we demonstrate that mESC-derived retinal tissue spontaneously acquires polarity reminiscent of the dorsal-ventral (D-V) patterning of the embryonic retina. Tbx5 and Vax2 were expressed in a mutually exclusive manner, as seen in vivo Three-dimensional morphometric analysis showed that the in vitro-formed optic cup often contains cleft structures resembling the embryonic optic fissure. To elucidate the mechanisms underlying the spontaneous D-V polarization of mESC-derived retina, we examined the effects of patterning factors, and found that endogenous BMP signaling plays a predominant role in the dorsal specification. Further analysis revealed that canonical Wnt signaling, which was spontaneously activated at the proximal region, acts upstream of BMP signaling for dorsal specification. These observations suggest that D-V polarity could be established within the self-formed retinal neuroepithelium by intrinsic mechanisms involving the spatiotemporal regulation of canonical Wnt and BMP signals.

Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration.

Retinal transplantation therapy for retinitis pigmentosa is increasingly of interest due to accumulating evidence of transplantation efficacy from animal studies and development of techniques for the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells into retinal tissues or cells. In this study, we aimed to assess the potential clinical utility of hESC-derived retinal tissues (hESC-retina) using newly developed primate models of retinal degeneration to obtain preparatory information regarding the potential clinical utility of these hESC-retinas in transplantation therapy. hESC-retinas were first transplanted subretinally into nude rats with or without retinal degeneration to confirm their competency as a graft to mature to form highly specified outer segment structure and to integrate after transplantation. Two focal selective photoreceptor degeneration models were then developed in monkeys by subretinal injection of cobalt chloride or 577-nm optically pumped semiconductor laser photocoagulation. The utility of the developed models and a practicality of visual acuity test developed for monkeys were evaluated. Finally, feasibility of hESC-retina transplantation was assessed in the developed monkey models under practical surgical procedure and postoperational examinations. Grafted hESC-retina was observed differentiating into a range of retinal cell types, including rod and cone photoreceptors that developed structured outer nuclear layers after transplantation. Further, immunohistochemical analyses suggested the formation of host-graft synaptic connections. The findings of this study demonstrate the clinical feasibility of hESC-retina transplantation and provide the practical tools for the optimization of transplantation strategies for future clinical applications.

Stem cells and genome editing: approaches to tissue regeneration and regenerative medicine.

Understanding the basis of regeneration of each tissue and organ, and incorporating this knowledge into clinical treatments for degenerative tissues and organs in patients, are major goals for researchers in regenerative biology. Here we provide an overview of current work, from high-regeneration animal models, to stem cell-based culture models, transplantation technologies, large-animal chimeric models, and programmable nuclease-based genome-editing technologies. Three-dimensional culture generating organoids, which represents intact tissue/organ identity including cell fate and morphology are getting more general approaches in the fields by taking advantage of embryonic stem cells, induced pluripotent stem cells and adult stem cells. The organoid culture system potentially has profound impact on the field of regenerative medicine. We also emphasize that the large animal model, in particular pig model would be a hope to manufacture humanized organs in in vivo empty (vacant) niche, which now potentially allows not only appropriate cell fate identity but nearly the same property as human organs in size. Therefore, integrative and collaborative researches across different fields might be critical to the aims needed in clinical trial.

Contractile actin belt and mesh structures provide the opposite dependence of epithelial stiffness on the spontaneous curvature of constituent cells.

Actomyosin generates contractile forces within cells, which have a crucial role in determining the macroscopic mechanical properties of epithelial tissues. Importantly, actin cytoskeleton, which propagates actomyosin contractile forces, forms several characteristic structures in a 3D intracellular space, such as a circumferential actin belt lining adherence junctions and an actin mesh beneath the apical membrane. However, little is known about how epithelial mechanical property depends on the intracellular contractile structures. We performed computational simulations using a 3D vertex model, and demonstrated the longitudinal tensile test of an epithelial tube, whose inside and outside are defined as the apical and basal surfaces, respectively. As a result, these subcellular structures provide the contrary dependence of epithelial stiffness and fracture force on the spontaneous curvature of constituent cells; the epithelial stiffness increases with increasing the spontaneous curvature in the case of belt, meanwhile it decreases in the case of mesh. This qualitative difference emerges from the different anisotropic deformability of apical cell surfaces; while belt preserves isotropic apical cell shapes, mesh does not. Moreover, the difference in the anisotropic deformability determines the frequency of cell rearrangements, which in turn effectively decrease the tube stiffness. These results illustrate the importance of the intracellular contractile structures, which may be regulated to optimize mechanical functions of individual epithelial tissues.

Self-organizing optic-cup morphogenesis in three-dimensional culture.

Balanced organogenesis requires the orchestration of multiple cellular interactions to create the collective cell behaviours that progressively shape developing tissues. It is currently unclear how individual, localized parts are able to coordinate with each other to develop a whole organ shape. Here we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse embryonic stem cell aggregates. Embryonic-stem-cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that became patterned along their proximal-distal axis. Whereas the proximal portion differentiated into mechanically rigid pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, as seen in vivo. We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulation of local epithelial properties.

Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex.

Here, using further optimized 3D culture that allows highly selective induction and long-term growth of human ES cell (hESC)-derived cortical neuroepithelium, we demonstrate unique aspects of self-organization in human neocorticogenesis. Self-organized cortical tissue spontaneously forms a polarity along the dorsocaudal-ventrorostral axis and undergoes region-specific rolling morphogenesis that generates a semispherical structure. The neuroepithelium self-forms a multilayered structure including three neuronal zones (subplate, cortical plate, and Cajal-Retzius cell zones) and three progenitor zones (ventricular, subventricular, and intermediate zones) in the same apical-basal order as seen in the human fetal cortex in the early second trimester. In the cortical plate, late-born neurons tend to localize more basally to early-born neurons, consistent with the inside-out pattern seen in vivo. Furthermore, the outer subventricular zone contains basal progenitors that share characteristics with outer radial glia abundantly found in the human, but not mouse, fetal brain. Thus, human neocorticogenesis involves intrinsic programs that enable the emergence of complex neocortical features.

In vitro organogenesis in three dimensions: self-organising stem cells.

Organ formation during embryogenesis is a complex process that involves various local cell-cell interactions at the molecular and mechanical levels. Despite this complexity, organogenesis can be modelled in vitro. In this article, we focus on two recent examples in which embryonic stem cells can self-organise into three-dimensional structures - the optic cup and the pituitary epithelium; and one case of self-organising adult stem cells - the gut epithelium. We summarise how these approaches have revealed intrinsic programs that drive locally autonomous modes of organogenesis and homeostasis. We also attempt to interpret the results of previous in vivo studies of retinal development in light of the self-organising nature of the retina.

Relaxation-expansion model for self-driven retinal morphogenesis: a hypothesis from the perspective of biosystems dynamics at the multi-cellular level.

The generation of complex organ structures such as the eye requires the intricate orchestration of multiple cellular interactions. In this paper, early retinal development is discussed with respect to the structure formation of the optic cup. Although recent studies have elucidated molecular mechanisms of retinal differentiation, little is known about how the unique shape of the optic cup is determined. A recent report has demonstrated that optic-cup morphogenesis spontaneously occurs in three-dimensional stem-cell culture without external forces, indicating a latent intrinsic order to generate the structure. Based on this self-organizing phenomenon, we introduce the "relaxation-expansion" model to mechanically interpret the tissue dynamics that enable the spontaneous invagination of the neural retina. This model involves three consecutive local rules (relaxation, apical constriction, and expansion), and its computer simulation recapitulates the optic-cup morphogenesis in silico.

In vitro induction of patterned branchial arch-like aggregate from human pluripotent stem cells.

Early patterning of neural crest cells (NCCs) in the craniofacial primordium is important for subsequent development of proper craniofacial structures. However, because of the complexity of the environment of developing tissues, surveying the early specification and patterning of NCCs is difficult. In this study, we develop a simplified in vitro 3D model using human pluripotent stem cells to analyze the early stages of facial development. In this model, cranial NCC-like cells spontaneously differentiate from neural plate border-like cells into maxillary arch-like mesenchyme after a long-term culture. Upon the addition of EDN1 and BMP4, these aggregates are converted into a mandibular arch-like state. Furthermore, temporary treatment with EDN1 and BMP4 induces the formation of spatially separated domains expressing mandibular and maxillary arch markers within a single aggregate. These results suggest that this in vitro model is useful for determining the mechanisms underlying cell fate specification and patterning during early facial development.

How might we build limbs in vitro informed by the modular aspects and tissue-dependency in limb development?

Building limb morphogenesis in vitro would substantially open up avenues for research and applications of appendage development. Recently, advances in stem cell engineering to differentiate desired cell types and produce multicellular structures in vitro have enabled the derivation of limb-like tissues from pluripotent stem cells. However, in vitro recapitulation of limb morphogenesis is yet to be achieved. To formulate a method of building limbs in vitro, it is critically important to understand developmental mechanisms, especially the modularity and the dependency of limb development on the external tissues, as those would help us to postulate what can be self-organized and what needs to be externally manipulated when reconstructing limb development in vitro. Although limbs are formed on the designated limb field on the flank of embryo in the normal developmental context, limbs can also be regenerated on the amputated stump in some animals and experimentally induced at ectopic locations, which highlights the modular aspects of limb morphogenesis. The forelimb-hindlimb identity and the dorsal-ventral, proximal-distal, and anterior-posterior axes are initially instructed by the body axis of the embryo, and maintained in the limb domain once established. In contrast, the aspects of dependency on the external tissues are especially underscored by the contribution of incoming tissues, such as muscles, blood vessels, and peripheral nerves, to developing limbs. Together, those developmental mechanisms explain how limb-like tissues could be derived from pluripotent stem cells. Prospectively, the higher complexity of limb morphologies is expected to be recapitulated by introducing the morphogen gradient and the incoming tissues in the culture environment. Those technological developments would dramatically enhance experimental accessibility and manipulability for elucidating the mechanisms of limb morphogenesis and interspecies differences. Furthermore, if human limb development can be modeled, drug development would be benefited by in vitro assessment of prenatal toxicity on congenital limb deficiencies. Ultimately, we might even create a future in which the lost appendage would be recovered by transplanting artificially grown human limbs.

RENGE infers gene regulatory networks using time-series single-cell RNA-seq data with CRISPR perturbations.

Single-cell RNA-seq analysis coupled with CRISPR-based perturbation has enabled the inference of gene regulatory networks with causal relationships. However, a snapshot of single-cell CRISPR data may not lead to an accurate inference, since a gene knockout can influence multi-layered downstream over time. Here, we developed RENGE, a computational method that infers gene regulatory networks using a time-series single-cell CRISPR dataset. RENGE models the propagation process of the effects elicited by a gene knockout on its regulatory network. It can distinguish between direct and indirect regulations, which allows for the inference of regulations by genes that are not knocked out. RENGE therefore outperforms current methods in the accuracy of inferring gene regulatory networks. When used on a dataset we derived from human-induced pluripotent stem cells, RENGE yielded a network consistent with multiple databases and literature. Accurate inference of gene regulatory networks by RENGE would enable the identification of key factors for various biological systems.

Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals.

During early telencephalic development, the major portion of the ventral telencephalic (subpallial) region becomes subdivided into three regions, the lateral (LGE), medial (MGE), and caudal (CGE) ganglionic eminences. In this study, we systematically recapitulated subpallial patterning in mouse embryonic stem cell (ESC) cultures and investigated temporal and combinatory actions of patterning signals. In serum-free floating culture, the dorsal-ventral specification of ESC-derived telencephalic neuroectoderm is dose-dependently directed by Sonic hedgehog (Shh) signaling. Early Shh treatment, even before the expression onset of Foxg1 (also Bf1; earliest marker of the telencephalic lineage), is critical for efficiently generating LGE progenitors, and continuous Shh signaling until day 9 is necessary to commit these cells to the LGE lineage. When induced under these conditions and purified by fluorescence-activated cell sorter, telencephalic cells efficiently differentiated into Nolz1(+)/Ctip2(+) LGE neuronal precursors and subsequently, both in culture and after in vivo grafting, into DARPP32(+) medium-sized spiny neurons. Purified telencephalic progenitors treated with high doses of the Hedgehog (Hh) agonist SAG (Smoothened agonist) differentiated into MGE- and CGE-like tissues. Interestingly, in addition to strong Hh signaling, the efficient specification of MGE cells requires Fgf8 signaling but is inhibited by treatment with Fgf15/19. In contrast, CGE differentiation is promoted by Fgf15/19 but suppressed by Fgf8, suggesting that specific Fgf signals play different, critical roles in the positional specification of ESC-derived ventral subpallial tissues. We discuss a model of the antagonistic Fgf8 and Fgf15/19 signaling in rostral-caudal subpallial patterning and compare it with the roles of these molecules in cortical patterning.

Delamination of trophoblast-like syncytia from the amniotic ectodermal analogue in human primed embryonic stem cell-based differentiation model.

Human primed embryonic stem cells (ESCs) are known to be converted to cells with several trophoblast properties, but it has remained controversial whether this phenomenon represents the inherent differentiation competence of human primed ESCs to trophoblast lineages. In this study, we report that chemical blockage of ACTIVIN/NODAL and FGF signals is sufficient to steer human primed ESCs into GATA3-expressing cells that give rise to placental hormone-producing syncytia analogous to syncytiotrophoblasts of the post-implantation stage of the human embryo. Despite their cytological similarity to syncytiotrophoblasts, these syncytia arise from the non-trophoblastic differentiation trajectory that recapitulates amniogenesis. These results provide insights into the possible extraembryonic differentiation pathway that is unique in primate embryogenesis.

Development of intensiometric indicators for visualizing N-cadherin interaction across cells.

N-cadherin (NCad) is a classical cadherin that mediates cell-cell interactions in a Ca2+-dependent manner. NCad participates in various biological processes, from ontogenesis to higher brain functions, though the visualization of NCad interactions in living cells remains limited. Here, we present intensiometric NCad interaction indicators, named INCIDERs, that utilize dimerization-dependent fluorescent proteins. INCIDERs successfully visualize reversible NCad interactions across cells. Compared to FRET-based indicators, INCIDERs have a ~70-fold higher signal contrast, enabling clear identification of NCad interactions. In primary neuronal cells, NCad interactions are visualized between closely apposed processes. Furthermore, visualization of NCad interaction at cell adhesion sites in dense cell populations is achieved by two-photon microscopy. INCIDERs are useful tools in the spatiotemporal investigation of NCad interactions across cells; future research should evaluate the potential of INCIDERs in mapping complex three-dimensional architectures in multi-cellular systems.

Optogenetic control of apical constriction induces synthetic morphogenesis in mammalian tissues.

The emerging field of synthetic developmental biology proposes bottom-up approaches to examine the contribution of each cellular process to complex morphogenesis. However, the shortage of tools to manipulate three-dimensional (3D) shapes of mammalian tissues hinders the progress of the field. Here we report the development of OptoShroom3, an optogenetic tool that achieves fast spatiotemporal control of apical constriction in mammalian epithelia. Activation of OptoShroom3 through illumination in an epithelial Madin-Darby Canine Kidney (MDCK) cell sheet reduces the apical surface of the stimulated cells and causes displacements in the adjacent regions. Light-induced apical constriction provokes the folding of epithelial cell colonies on soft gels. Its application to murine and human neural organoids leads to thickening of neuroepithelia, apical lumen reduction in optic vesicles, and flattening in neuroectodermal tissues. These results show that spatiotemporal control of apical constriction can trigger several types of 3D deformation depending on the initial tissue context.

Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation.

Embryonic stem (ES) cells differentiate into neuroectodermal progenitors when cultured as floating aggregates in serum-free conditions. Here, we show that strict removal of exogenous patterning factors during early differentiation steps induces efficient generation of rostral hypothalamic-like progenitors (Rax(+)/Six3(+)/Vax1(+)) in mouse ES cell-derived neuroectodermal cells. The use of growth factor-free chemically defined medium is critical and even the presence of exogenous insulin, which is commonly used in cell culture, strongly inhibits the differentiation via the Akt-dependent pathway. The ES cell-derived Rax(+) progenitors generate Otp(+)/Brn2(+) neuronal precursors (characteristic of rostral-dorsal hypothalamic neurons) and subsequently magnocellular vasopressinergic neurons that efficiently release the hormone upon stimulation. Differentiation markers of rostral-ventral hypothalamic precursors and neurons are induced from ES cell-derived Rax(+) progenitors by treatment with Shh. Thus, in the absence of exogenous growth factors in medium, the ES cell-derived neuroectodermal cells spontaneously differentiate into rostral (particularly rostral-dorsal) hypothalamic-like progenitors, which generate characteristic hypothalamic neuroendocrine neurons in a stepwise fashion, as observed in vivo. These findings indicate that, instead of the addition of inductive signals, minimization of exogenous patterning signaling plays a key role in rostral hypothalamic specification of neural progenitors derived from pluripotent cells.