Neural rosette formation is a critical morphogenetic process during neural development, whereby neural stem cells are enclosed in rosette niches to equipoise proliferation and differentiation. How neural rosettes form and provide a regulatory micro-environment remains to be elucidated. We employed the human embryonic stem cell-based neural rosette system to investigate the structural development and function of neural rosettes. Our study shows that neural rosette formation consists of five types of morphological change: intercalation, constriction, polarization, elongation and lumen formation. Ca2+ signaling plays a pivotal role in the five steps by regulating the actions of the cytoskeletal complexes, actin, myosin II and tubulin during intercalation, constriction and elongation. These, in turn, control the polarizing elements, ZO-1, PARD3 and β-catenin during polarization and lumen production for neural rosette formation. We further demonstrate that the dismantlement of neural rosettes, mediated by the destruction of cytoskeletal elements, promotes neurogenesis and astrogenesis prematurely, indicating that an intact rosette structure is essential for orderly neural development.
- MeSH
- aktiny metabolismus MeSH
- apoptóza účinky léků MeSH
- buněčný rodokmen účinky léků MeSH
- cytoskelet účinky léků metabolismus MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie účinky léků metabolismus MeSH
- myosin typu II metabolismus MeSH
- nervové kmenové buňky cytologie účinky léků metabolismus MeSH
- neurogeneze účinky léků MeSH
- neurony cytologie účinky léků metabolismus ultrastruktura MeSH
- polarita buněk účinky léků MeSH
- protein zonula occludens 1 metabolismus MeSH
- tvar buňky * účinky léků MeSH
- tvorba rozet * MeSH
- vápník farmakologie MeSH
- vápníková signalizace * účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Human pluripotent stem cells (hPSC) require signaling provided by fibroblast growth factor (FGF) receptors. This can be initiated by the recombinant FGF2 ligand supplied exogenously, but hPSC further support their niche by secretion of endogenous FGF2. In this study, we describe a role of tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase in this process. We show that TEC-mediated FGF2 secretion is essential for hPSC self-renewal, and its lack mediates specific differentiation. Following both short hairpin RNA- and small interfering RNA-mediated TEC knockdown, hPSC secretes less FGF2. This impairs hPSC proliferation that can be rescued by increasing amounts of recombinant FGF2. TEC downregulation further leads to a lower expression of the pluripotency markers, an improved priming towards neuroectodermal lineage, and a failure to develop cardiac mesoderm. Our data thus demonstrate that TEC is yet another regulator of FGF2-mediated hPSC pluripotency and differentiation. Stem Cells 2017;35:2050-2059.
- MeSH
- biologické markery metabolismus MeSH
- buněčné linie MeSH
- buněčný rodokmen * účinky léků MeSH
- down regulace účinky léků MeSH
- fibroblastový růstový faktor 2 metabolismus sekrece MeSH
- lidé MeSH
- pluripotentní kmenové buňky cytologie enzymologie MeSH
- proliferace buněk účinky léků MeSH
- rekombinantní proteiny farmakologie MeSH
- tyrosinkinasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
During embryogenesis, the Activin/Nodal pathway promotes the mesendodermal lineage and inhibits neural fate. The molecular mechanisms underlying this role of the Activin/Nodal pathway are not clear. In this study, we report a role for protein tyrosine phosphatase 1B (PTP1B) in Activin-mediated early fate decisions during ESC differentiation and show that PTP1B acts as an effector of the Activin pathway to specify mesendodermal or neural fate. We found that the Activin/ALK4 pathway directly recruits PTP1B and stimulates its release from the endoplasmic reticulum through ALK4-mediated cleavage. Subsequently, PTP1B suppresses p-ERK1/2 signaling to inhibit neural specification and promote mesendodermal commitment. These findings suggest that a noncanonical Activin signaling pathway functions in lineage specification of mouse and human embryonic stem cells.
- MeSH
- aktivinové receptory typu I chemie metabolismus MeSH
- aktiviny metabolismus MeSH
- benzamidy farmakologie MeSH
- buněčná diferenciace * účinky léků MeSH
- buněčný rodokmen účinky léků MeSH
- dioxoly farmakologie MeSH
- embryonální kmenové buňky cytologie účinky léků enzymologie MeSH
- endoderm cytologie účinky léků metabolismus MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- fosforylace účinky léků MeSH
- lidé MeSH
- MAP kinasový signální systém účinky léků MeSH
- mezoderm cytologie účinky léků metabolismus MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- neurony cytologie účinky léků metabolismus MeSH
- pluripotentní kmenové buňky cytologie účinky léků metabolismus MeSH
- protein Smad2 metabolismus MeSH
- sekvence aminokyselin MeSH
- signální transdukce * účinky léků MeSH
- tyrosinfosfatasa nereceptorového typu 1 metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We isolated and expanded stem cells from dental pulp from extracted third molars using an innovative culture method consisting of low serum-containing medium supplemented with epidermal growth factor and platelet-derived growth factor BB. We evaluated the differentiation potential of these cells when they were growing either adherently or as micromass/spheroid cultures in various media. Undifferentiated and differentiated cells were analyzed by flow cytometry, immunocytochemistry and immunoblotting. The flow cytometry results showed that the dental pulp stem cells (DPSCs) were positive for mesenchymal stromal cell markers, but negative for hematopoietic markers. Immunocytochemical and/or immunoblotting analyses revealed the expression of numerous stem cell markers, including nanog, Sox2, nestin, Musashi-1 and nucleostemin, whereas they were negative for markers associated with differentiated neural, vascular and hepatic cells. Surprisingly, the cells were only slightly positive for α-smooth muscle actin, and a heterogeneous expression of CD146 was observed. When cultured in osteogenic media, they expressed osteonectin, osteopontin and procollagen I, and in micromass cultures, they produced collagen I. DPSCs cultured in TGF-β1/3-supplemented media produced extracellular matrix typical of cartilaginous tissue. The addition of vascular endothelial growth factor to serum-free media resulted in the expression of endothelial markers. Interestingly, when cultured in neurogenic media, DPSCs exhibited de novo or upregulated markers of undifferentiated and differentiated neural cells. Collectively, our data show that DPSCs are self-renewing and able to express markers of bone, cartilage, vascular and neural tissues, suggesting their multipotential capacity. Their easy accessibility makes these cells a suitable source of somatic stem cells for tissue engineering.
- MeSH
- aktiny metabolismus MeSH
- antigen CD146 metabolismus MeSH
- buněčná diferenciace účinky léků MeSH
- buněčný rodokmen účinky léků MeSH
- chondrogeneze účinky léků MeSH
- endoteliální buňky cytologie účinky léků metabolismus MeSH
- imunohistochemie MeSH
- kmenové buňky účinky léků patologie MeSH
- kultivační média bez séra farmakologie MeSH
- kultivované buňky MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- molár třetí patologie MeSH
- myocyty hladké svaloviny cytologie účinky léků metabolismus MeSH
- myofibroblasty cytologie účinky léků metabolismus MeSH
- neurony cytologie účinky léků metabolismus MeSH
- osteogeneze účinky léků MeSH
- průtoková cytometrie MeSH
- zaklíněný zub patologie MeSH
- zubní dřeň patologie MeSH
- Check Tag
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
