Project description:During cerebellar development, the main portion of the cerebellar plate neuroepithelium (NE) gives birth to Purkinje cells and interneurons, while the germinal zone at its dorsal edge, called the rhombic lip (RL), generates granule cells and cerebellar nuclei neurons. However, it remains elusive how these components work together to generate the intricate structure of the cerebellar anlage. In this study, we found that a polarized cerebellar anlage structure self-organizes in three-dimensional (3D) human ES cell (hESC) culture. This NE is capable of differentiating into electrophysiologically functional Purkinje cells. The addition of FGF19 promotes spontaneous generation of dorsoventrally polarized NE structures containing cerebellar and basal plates. Furthermore, further addition of SDF1 promoted the generation of stratified cerebellar plate NE with RL-like germinal zones self-forming at the edge. Thus, hESC-derived cerebellar progenitors exhibit substantial self-organizing potential for generating a polarized structure reminiscent of the early human cerebellar anlage at the first trimester. Examination of mRNA profile in two different treated human ES cells .
Project description:During cerebellar development, the main portion of the cerebellar plate neuroepithelium (NE) gives birth to Purkinje cells and interneurons, while the germinal zone at its dorsal edge, called the rhombic lip (RL), generates granule cells and cerebellar nuclei neurons. However, it remains elusive how these components work together to generate the intricate structure of the cerebellar anlage. In this study, we found that a polarized cerebellar anlage structure self-organizes in three-dimensional (3D) human ES cell (hESC) culture. This NE is capable of differentiating into electrophysiologically functional Purkinje cells. The addition of FGF19 promotes spontaneous generation of dorsoventrally polarized NE structures containing cerebellar and basal plates. Furthermore, further addition of SDF1 promoted the generation of stratified cerebellar plate NE with RL-like germinal zones self-forming at the edge. Thus, hESC-derived cerebellar progenitors exhibit substantial self-organizing potential for generating a polarized structure reminiscent of the early human cerebellar anlage at the first trimester.
Project description:Transcription profiling by high throughput sequencing of human polarized cerebellar plate neuroepithelium in three-dimensional culture
Project description:All mRNA was isolated after 8 hours of culture time in each of three culture conditions. (1) TCPS Plate, (2) Collagen-GAG 2 dimensional coated plate and (3) collagen-GAG three dimensional mesh. Keywords: ordered
Project description:All mRNA was isolated after 8 hours of culture time in each of three culture conditions. (1) TCPS Plate, (2) Collagen-GAG 2 dimensional coated plate and (3) collagen-GAG three dimensional mesh.
Project description:Pluripotent stem cells can be differentiated into three-dimensional (3D) retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall bioreactors (RWB) to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWB demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWB organoids at day (D)25 reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.
Project description:Directed differentiation methods allow acquisition of high-purity cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs); however, their immaturity characteristic limits their application for drug screening and regenerative therapy. The rapid electrical pacing of cardiomyocytes have been used for efficiently promoting the maturation of cardiomyocytes, here we describe a simple device in modified culture plate on which hiPSC-derived CMs can form three-dimensional self-organized tissue rings (SOTRs). Using calcium imaging, we show that within the ring, traveling waves (TWs) of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz. After 2 weeks, SOTRs with TW training show matured features including structural organization, increased cardiac-specific gene expression, enhanced Ca2+-handling properties, an increased oxygen-consumption rate, and enhanced contractile force. We subsequently use a mathematical model to interpret the origination, propagation, and long-term behavior of the TWs within the SOTRs. The TW could also potentially be used for pacing the electrical excitable cells such as neuron and retina cells for various applications.
Project description:Pluripotent stem cells (PSC) can differentiate inot any cell type of an organism. Their remarkable capability of self-organization enables the formation of three-dimensional structures that resembles miniature organs, including cerebral organoids. These organoids can recreate early steps of the human cerebral cortex development, and show great potential for modeling human diseases, particularly for those with a developmental component. This data evidences stem cell-derived cerebral organoids as a key model to study brain development and neurodevelopmental, neurodegenerative and neuropsychiatric diseases.
Project description:Differentiation of monolayered epithelia is characterized by the formation of a basoapical polarity axis, except during the early stages of cancer development. Using mammary glandular structures (acini) produced in a three-dimensional cell culture system we have demonstrated that, in order for mammary epithelial cells to exit quiescence and enter the cell cycle, acini have to lose apical polarity. In order to identify the genes dependent on apical polarity that could control cell quiescence, and possibly other aspects of tissue homeostasis, we have used Affymetrix technology microarray analysis of the 22,277 features/genes of the Human Genome U133A 2.0 array Chip in apically polarized and non-polarized breast epithelial acinar cells in three-dimensional culture. Genes commonly down-regulated in two treatments that altered apical polarity compared to control were considered to be dependent on apical polarity status for their transcription. Apically polarized (Control) and two different populations of non-polarized human mammary epithelial cells (18-alpha-glycyrrhetinic acid =AGA, and 5-aza-2'-deoxycytidine =Aza) were used for microarray analysis containing four biological replicates (1-4) for each treatment. The two treatments that cause loss of apical polarity were compared to reference apically polarized control samples using two different statistical analysis methods (FDR and HolmM-bM-^@M-^Ys).
Project description:Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human induced pluripotent stem cells (hiPSCs). Toward clinical applications on hPSC-derived retinal sheets, the establishment of a quality control (QC) strategy for 3D-retinas and dissected retinal sheets has remained a major challenge. We performed a microarray analysis for retinal tissue and off-target tissue to identify the major off-target tissue in hiPSC-culture.