[Show abstract][Hide abstract] ABSTRACT: Studies have provided important findings about the roles of Notch signaling in neural development. Unfortunately, however, most of these studies have investigated the neural stem cells (NSCs) of mice or other laboratory animals rather than humans, mainly owing to the difficulties associated with obtaining human brain samples. It prompted us to focus on neuroectodermal spheres (NESs) which are derived from human embryonic stem cell (hESC) and densely inhabited by NSCs. We here investigated the role of Notch signaling with the hESC-derived NESs.
From hESCs, we derived NESs, the in-vitro version of brain-derived neurospheres. NES formation was confirmed by increased levels of various NSC marker genes and the emergence of rosette structures in which neuroprogenitors are known to reside. We found that Notch signaling, which maintains stem cell characteristics of in-vivo-derived neuroprogenitors, is active in these hESC-derived NESs, similar to their in-vivo counterpart. Expression levels of Notch signaling molecules such as NICD, DLLs, JAG1, HES1 and HES5 were increased in the NESs. Inhibition of the Notch signaling by a gamma-secretase inhibitor reduced rosette structures, expression levels of NSC marker genes and proliferation potential in the NESs, and, if combined with withdrawal of growth factors, triggered differentiation toward neurons.
Our results indicate that the hESC-derived NESs, which share biochemical features with brain-derived neurospheres, maintain stem cell characteristics mainly through Notch signaling, which suggests that the hESC-derived NESs could be an in-vitro model for in-vivo neurogenesis.
[Show abstract][Hide abstract] ABSTRACT: The ability to generate neural lineages from human embryonic stem cells (hESCs) in a controlled manner would further investigation of human neurogenesis and development of potential cell therapeutic applications to treat neurological diseases; however, generating such neural stem cells (NSCs) remains a challenge. In an attempt to characterize the cellular mechanisms involved in hESC differentiation into neuroprogenitor cells, we performed 2-DE using protein extracts from hESC-derived embryoid bodies (EBs) and neuroectodermal spheres (NESs) bearing neuroprogenitors. Of 47 differentially expressed protein spots, 28 nonredundant spots were shown to be upregulated in the NESs; these protein spots included neurogenesis-related proteins (TAF1, SEPT2, NPH3, and CRABP), as expected. Interestingly, 6 of these 28 protein spots were cytoskeleton-associated proteins (CSAP) such as Fascin-1, Cofilin-1, and Stathmin-1. Western-blot analyses confirmed the increased levels of these proteins in the NESs. Furthermore, immunostaining analysis showed that both Fascin-1 and Stathmin-1 were preferentially expressed in the inner rims of neural rosettes, which are characteristic features of neuroprogenitors in culture. We also confirmed prominent expression of Fascin-1 in (sub-)ventricular zone in E15.5 mouse fetal brain. Our results suggest that, in addition to the induction of those genes involved in neural development, hESC differentiation into the NES is associated with a marked reorganization of the cellular cytoskeleton.
[Show abstract][Hide abstract] ABSTRACT: Complex signaling pathways operate in human embryonic stem cells (hESCs) and are coordinated to maintain self-renewal and stem cell characteristics in them. Protein tyrosine kinases (PTKs) participate in diverse signaling pathways in various types of cells. Because of their functions as key molecules in various cellular processes, PTKs are anticipated to have important roles also in hESCs. In this study, we investigated the roles of PTKs in undifferentiated and differentiated hESCs. To establish comprehensive PTK expression profiles in hESCs, we performed reverse transcriptase PCR using degenerate primers according to the conserved catalytic PTK motifs in both undifferentiated and differentiated hESCs. Here, we identified 42 different kinases in two hESC lines, including 5 non-receptor tyrosine kinases (RTKs), 24 RTKs, and 13 dual and other kinases, and compared the protein kinase expression profiles of hESCs and retinoic acid-treated hESCs. Significantly, up- and downregulated kinases in undifferentiated hESCs were confirmed by real-time PCR and western blotting. MAP3K3, ERBB2, FGFR4, and EPHB2 were predominantly upregulated, while CSF1R, TYRO3, SRC, and GSK3A were consistently downregulated in two hESC lines. Western blot analysis showed that the transcriptional levels of these kinases were consistent with the translational levels. The obstruction of upregulated kinases' activities using specific inhibitors disturbed the undifferentiated status and induced the differentiation of hESCs. Our results support the dynamic expression of PTKs during hESC maintenance and suggest that specific PTKs that are consistently up- and downregulated play important roles in the maintenance of stemness and the direction of differentiation of hESCs.
[Show abstract][Hide abstract] ABSTRACT: We have developed a novel method for fabricating an embryonic stem cell divider (ESCD) constructed from a poly(dimethylsiloxane) (PDMS) replica with a square or hexagonal pattern, and have proposed a new dissociation method for human embryonic stem cells (ESCs). An aspect ratio of the device as high as 2 was perfectly replicated in the cutting line. Using the ESCD, human ESC colonies can be easily and efficiently dissociated into regular-sized ESC clumps without enzymatic treatment. The regularity of the ESC clumps dissociated by the ESCD was compared to that dissociated by a conventional mechanical method. Its quality and reliability were confirmed by maintaining undifferentiated ESCs up to the 15th passage. The ESCD will contribute to the advance quality control of in vitro ESC cultures and allow large-scale production of qualified ESCs with tremendous time- and work-saving.