RESUMO
Salivary glands are exocrine glands composed of several cell types, including the ductal, acinar, and basal/myoepithelial cells. They play important roles in maintaining oral homeostasis and health. During early murine development, the salivary glands, which arise as epithelial buds, are produced from primitive oral epithelia through an interaction between the oral epithelium and mesenchyme.We recently reported that salivary gland organoids can be generated from mouse embryonic stem cells (ESCs). We recapitulated the process of embryonic salivary gland development using an organoid culture system. The mouse ESC-derived salivary gland organoids consisted of acinar-, ductal-, and myoepithelial-like cells. In this chapter, we describe a protocol for differentiating salivary gland organoids from ESCs .
Assuntos
Células-Tronco Embrionárias Murinas , Organoides , Animais , Mesoderma , Camundongos , Organogênese , Organoides/metabolismo , Glândulas SalivaresRESUMO
Humans have a large and gyrencephalic brain. The higher intellectual ability of humans is dependent on the proper development of the brain. Brain malformation is often associated with cognitive dysfunction. It is thus important to know how our brain grows during development. Several animal species have been used as models to understand the mechanisms of brain development, and have provided us with basic information in this regard. It has been revealed that mammalian brain development basically proceeds through a similar process by common mechanisms, including neural stem cell proliferation and neurogenesis. However, humans also display species-specific features in these processes. These differences seem to be important for building the proper human brain structure. Analysis of these human-specific features requires human brain samples, which are difficult to obtain due to both ethical and practical reasons. Nevertheless, brain organoids derived from human pluripotent stem cells can be used as models to study human brain development and pathology because such organoids can partly recapitulate human fetal developmental processes. In this review, we will review some human-specific features during brain development and discuss brain organoid technology as a model system. We will especially focusing on neocortical development.
Assuntos
Modelos Biológicos , Neocórtex/crescimento & desenvolvimento , Organoides/fisiologia , Animais , Humanos , Especificidade da Espécie , Técnicas de Cultura de Tecidos/métodosRESUMO
In the developing embryo, telencephalon arises from the rostral portion of the neural tube. The telencephalon further subdivides into distinct brain regions along the dorsal-ventral (DV) axis by exogenous patterning signals. Here, we describe a protocol for in vitro generation of various telencephalic regions from human embryonic stem cells (ESCs). Dissociated human ESCs are reaggregated in a low-cell-adhesion 96-well plate and cultured as floating aggregates. Telencephalic neural progenitors are efficiently generated when ESC aggregates are cultured in serum-free medium containing TGFß inhibitor and Wnt inhibitor. In long-term culture, the telencephalic neural progenitors acquire cortical identities and self-organize a stratified cortical structure as seen in human fetal cortex. By treatment with Shh signal, the telencephalic progenitors acquire ventral (subpallial) identities and generate lateral ganglionic eminence (LGE) and medial ganglionic eminence (MGE). In contrast, by treatment with Wnt and BMP signals, their regional identities shift to more dorsal side that generates choroid plexus and medial palllium (hippocampal primordium).
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Telencéfalo/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos/citologia , Cistos Glanglionares/fisiopatologia , Hipocampo/citologia , HumanosRESUMO
A three-dimensional (3D) tissue generated in vitro is a promising source to study developmental biology and regenerative medicine. In the last decade, Yoshiki Sasai's group have developed a 3D stem cell culture technique known as SFEBq and demonstrated that embryonic stem cells (ESCs) have an ability to self-organize stratified neural tissue including 3D-retina. Furthermore, we have reported that ESC-derived retinal tissue can form an optic cup and a ciliary margin, which are unique structures in the developing retina. In this review, we focus on self-organizing culture technique to generate 3D-retina from human ESCs.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Retina/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , CamundongosRESUMO
The thalamus is a diencephalic structure that plays crucial roles in relaying and modulating sensory and motor information to the neocortex. The thalamus develops in the dorsal part of the neural tube at the level of the caudal forebrain. However, the molecular mechanisms that are essential for thalamic differentiation are still unknown. Here, we have succeeded in generating thalamic neurons from mouse embryonic stem cells (mESCs) by modifying the default method that induces the most-anterior neural type in self-organizing culture. A low concentration of the caudalizing factor insulin and a MAPK/ERK kinase inhibitor enhanced the expression of the caudal forebrain markers Otx2 and Pax6. BMP7 promoted an increase in thalamic precursors such as Tcf7l2+/Gbx2+ and Tcf7l2+/Olig3+ cells. mESC thalamic precursors began to express the glutamate transporter vGlut2 and the axon-specific marker VGF, similar to mature projection neurons. The mESC thalamic neurons extended their axons to cortical layers in both organotypic culture and subcortical transplantation. Thus, we have identified the minimum elements sufficient for in vitro generation of thalamic neurons. These findings expand our knowledge of thalamic development.