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The neuroectoderm is patterned along a rostral-caudal axis in response to localized factors in the embryo, but exactly how these factors act as positional information for this patterning is not yet fully understood. Here, using the self-organizing properties of mouse embryonic stem cell (ESC), we report that ESC-derived neuroectoderm self-generates a Six3+ rostral and a Irx3+ caudal bipolarized patterning. In this instance, localized Fgf signaling performs dual roles, as it regulates Six3+ rostral polarization at an earlier stage and promotes Wnt signaling at a later stage. The Wnt signaling components are differentially expressed in the polarized tissues, leading to genome-wide Irx3+ caudal-polarization signals. Surprisingly, differentially expressed Wnt agonists and antagonists have essential roles in orchestrating the formation of a balanced rostral-caudal neuroectoderm pattern. Together, our findings provide key processes for dynamic self-patterning and evidence that a temporally and locally regulated interaction between Fgf and Wnt signaling controls self-patterning in ESC-derived neuroectoderm.

Pluripotent stem cells can undergo repeated self-renewal while retaining genetic integrity, but they occasionally acquire aneuploidy during long-term culture, which is a practical obstacle for medical applications of human pluripotent stem cells. In this study, we explored the biological roles of ABR, a regulator of RHO family small GTPases, and found that it has pivotal roles during mitotic processes in human embryonic stem cells (hESCs). Although ABR has been shown to be involved in dissociation-induced hESC apoptosis, it does not appear to have direct effects on cell survival unless cell-cell contact is impaired. Instead, we found that it is important for faithful hESC division. Mechanistically, ABR depletion compromised centrosome dynamics and predisposed the cell to chromosome misalignment and missegregation, which raised the frequency of aneuploidy. These results provide insights into the mechanisms that support the genetic integrity of self-renewing hESCs.

Morphogenesis in multicellular organisms is accompanied by apoptotic cell behaviors: cell shrinkage and cell disappearance. The mechanical effects of these behaviors are spatiotemporally regulated within multicellular dynamics to achieve proper tissue sizes and shapes in three-dimensional (3D) space. To analyze 3D multicellular dynamics, 3D vertex models have been suggested, in which a reversible network reconnection (RNR) model has successfully expressed 3D cell rearrangements during large deformations. To analyze the effects of apoptotic cell behaviors on 3D multicellular morphogenesis, we modeled cell apoptosis based on the RNR model framework. Cell shrinkage was modeled by the potential energy as a function of individual cell times during the apoptotic phase. Cell disappearance was modeled by merging neighboring polyhedrons at their boundary surface according to the topological rules of the RNR model. To establish that the apoptotic cell behaviors could be expressed as modeled, we simulated morphogenesis driven by cell apoptosis in two types of tissue topology: 3D monolayer cell sheet and 3D compacted cell aggregate. In both types of tissue topology, the numerical simulations successfully illustrated that cell aggregates gradually shrank because of successive cell apoptosis. During tissue shrinkage, the number of cells in aggregates decreased while maintaining individual cell size and shape. Moreover, in case of localizing apoptotic cells within a part of the 3D monolayer cell aggregate, the cell apoptosis caused the global tissue bending by pulling on surrounding cells. In case of localizing apoptotic cells on the surface of the 3D compacted cell aggregate, the cell apoptosis caused successive, directional cell rearrangements from the inside to the surface. Thus, the proposed model successfully provided a basis for expressing apoptotic cell behaviors during 3D multicellular morphogenesis based on an RNR model framework.

Anterior pituitary is critical for endocrine systems. Its hormonal responses to positive and negative regulators are indispensable for homeostasis. For this reason, generating human anterior pituitary tissue that retains regulatory hormonal control in vitro is an important step for the development of cell transplantation therapy for pituitary diseases. Here we achieve this by recapitulating mouse pituitary development using human embryonic stem cells. We find that anterior pituitary self-forms in vitro following the co-induction of hypothalamic and oral ectoderm. The juxtaposition of these tissues facilitated the formation of pituitary placode, which subsequently differentiated into pituitary hormone-producing cells. They responded normally to both releasing and feedback signals. In addition, after transplantation into hypopituitary mice, the in vitro-generated corticotrophs rescued physical activity levels and survival of the hosts. Thus, we report a useful methodology for the production of regulator-responsive human pituitary tissue that may benefit future studies in regenerative medicine.

The developing dorsomedial telencephalon includes the medial pallium, which goes on to form the hippocampus. Generating a reliable source of human hippocampal tissue is an important step for cell-based research into hippocampus-related diseases. Here we show the generation of functional hippocampal granule- and pyramidal-like neurons from self-organizing dorsomedial telencephalic tissue using human embryonic stem cells (hESCs). First, we develop a hESC culture method that utilizes bone morphogenetic protein (BMP) and Wnt signalling to induce choroid plexus, the most dorsomedial portion of the telencephalon. Then, we find that titrating BMP and Wnt exposure allowed the self-organization of medial pallium tissues. Following long-term dissociation culture, these dorsomedial telencephalic tissues give rise to Zbtb20+/Prox1+ granule neurons and Zbtb20+/KA1+ pyramidal neurons, both of which were electrically functional with network formation. Thus, we have developed an in vitro model that recapitulates human hippocampus development, allowing the generation of functional hippocampal granule- and pyramidal-like neurons.

Tissue morphogenesis in multicellular organisms is accompanied by proliferative cell behaviors: cell division (increase in cell number after each cell cycle) and cell growth (increase in cell volume during each cell cycle). These proliferative cell behaviors can be regulated by multicellular dynamics to achieve proper tissue sizes and shapes in three-dimensional (3D) space. To analyze multicellular dynamics, a reversible network reconnection (RNR) model has been suggested, in which each cell shape is expressed by a single polyhedron. In this study, to apply the RNR model to simulate tissue morphogenesis involving proliferative cell behaviors, we model cell proliferation based on a RNR model framework. In this model, cell division was expressed by dividing a polyhedron at a planar surface for which cell division behaviors were characterized by three quantities: timing, intracellular position, and normal direction of the dividing plane. In addition, cell growth was expressed by volume growth as a function of individual cell times within their respective cell cycles. Numerical simulations using the proposed model showed that tissues grew during successive cell divisions with several cell cycle times. During these processes, the cell number in tissues increased while maintaining individual cell size and shape. Furthermore, tissue morphology dramatically changed based on different regulations of cell division directions. Thus, the proposed model successfully provided a basis for expressing proliferative cell behaviors during morphogenesis based on a RNR model framework.

In general, a rapidly growing epithelial sheet during tissue morphogenesis shows a smooth and continuous curvature on both inner cavity (apical) and basement membrane (basal) sides. For instance, epithelia of the neural tube and optic vesicle in the early embryo maintain continuous curvatures in their local domains, even during their rapid growth. However, given that cell divisions, which substantially perturb the local force balance, frequently and successively occur in an uncoordinated manner, it is not self-evident to explain how the tissue keeps a continuous curvature at large. In the majority of developing embryonic epithelia with smooth surfaces, their curvatures are apically concave, because of the presence of strong tangential contractile force on the apical side. In this numerical study, we demonstrate that tangential contractile forces on the apical surface play a critical role in the maintenance of smooth curvatures in the epithelium and reduce irregular undulations caused by uncoordinated generation of local pushing force. Using a reversible network reconnection (RNR) model, which we previously developed to make numerical analyses highly reproducible even under rapid tissue-growth conditions, we performed simulations for morphodynamics to examine the effect of apical contractile forces on the continuity of curvatures. Interestingly, the presence of apical contractile forces suppressed irregular undulations not only on the apical side but also on the basal surface. These results indicate that cellular contractile forces on the apical surface control not only the shape at a single cell level but also at a tissue level as a result of emergent mechanical coordination.

Morphogenesis of tissues in organ development is accompanied by large three-dimensional (3D) deformations, in which mechanical interactions among multiple cells are spatiotemporally regulated. To reveal the deformation mechanisms, in this study, we developed the reversible network reconnection (RNR) model. The model is developed on the basis of 3D vertex model, which expresses a multicellular aggregate as a network composed of vertices. 3D vertex models have successfully simulated morphogenetic dynamics by expressing cellular rearrangements as network reconnections. However, the network reconnections in 3D vertex models can cause geometrical irreversibility, energetic inconsistency, and topological irreversibility, therefore inducing unphysical results and failures in simulating large deformations. To resolve these problems, we introduced (1) a new definition of the shapes of polygonal faces between cellular polyhedrons, (2) an improved condition for network reconnections, (3) a new condition for potential energy functions, and (4) a new constraint condition for the shapes of polygonal faces that represent cell–cell boundaries. Mathematical and computational analyses demonstrated that geometrical irreversibility, energetic inconsistency, and topological irreversibility were resolved by suppressing the geometrical gaps in the network and avoiding the generation of irreversible network patterns in reconnections. Lastly, to demonstrate the applicability of the RNR model, we simulated tissue deformation of growing cell sheets and showed that our model can simulate large tissue deformations, in which large changes occur in the local curvatures and layer formations of tissues. Thus, the RNR model enables in silico recapitulation of complex tissue morphogenesis.

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6(+) cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6(+) cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP(+) cells, while enhancer 1056 is active in Olig2(+) cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6(+) cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6(+) cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP(+) cells, while enhancer 1056 is active in Olig2(+) cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.

Directed differentiation from human pluripotent stem cells (hPSCs) has seen significant progress in recent years. However, most differentiated populations exhibit immature properties of an early embryonic stage, raising concerns about their ability to model and treat disease. Here, we report the directed differentiation of hPSCs into medial ganglionic eminence (MGE)-like progenitors and their maturation into forebrain type interneurons. We find that early-stage progenitors progress via a radial glial-like stem cell enriched in the human fetal brain. Both in vitro and posttransplantation into the rodent cortex, the MGE-like cells develop into GABAergic interneuron subtypes with mature physiological properties along a prolonged intrinsic timeline of up to 7 months, mimicking endogenous human neural development. MGE-derived cortical interneuron deficiencies are implicated in a broad range of neurodevelopmental and degenerative disorders, highlighting the importance of these results for modeling human neural development and disease.

Nasu M, Takata N, Danjo T, Sakaguchi H, Kadoshima T, Futaki S, et al. (2012) Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture. PLoS ONE 7(12): e53024. https://doi.org/10.1371/journal.pone.0053024, In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development.

Nasu M, Takata N, Danjo T, Sakaguchi H, Kadoshima T, Futaki S, et al. (2012) Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture. PLoS ONE 7(12): e53024. https://doi.org/10.1371/journal.pone.0053024, In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development.