Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferati...Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferative regeneration. However, substantial proliferation occurs in organotypic cultures of cochleae from postnatal mice. In the present study, we studied the time course of proliferative growth in cultures of mouse cochlea explants obtained from up to 12 postnatal days. The mitotic nature of this growth was confirmed by bromodeoxyuridine (BrdU) staining and expression of proliferation cell nuclear antigen (PCNA) evaluated with real-time quantitative poly-merase chain reaction(RT-PCR). Similar growth time course was found in the cochlear explants of different postnatal ages. The new growth reached its maximum at around 2 days in culture followed by a slow-down, and virtually stopped after 5 days of culture. The possible mechanisms and the significance of this proliferation are discussed.展开更多
BACKGROUND Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss.Cells extracted from peripheral corneas can form stem cell-enriched s...BACKGROUND Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss.Cells extracted from peripheral corneas can form stem cell-enriched spheres,which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic tissue.AIM To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic tissue.METHODS Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal cells.These spheres were implanted into incisions created in full thickness and onto the surface of 10μm thin sections of keratoconic and normal stromal tissues in vitro.Tissue sections were used to maximise use of limited keratoconic tissue available for research.Living cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d(D14)from implantation.Sphere cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus treatment.RESULTS Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10μm thin en face tissue sections.No observable differences were seen in sphere migration,proliferation or differentiation in keratoconic tissue compared to normal between day 0 and D14.Spheres stained positively with Calcein-AM up to D14.Cell migration increased from day 0 to D14,occurring radially in three dimensions from the sphere and in alignment with tissue edges.Cell proliferation marker,EdU,was detected at day 10.Implanted spheres stained positively for putative stem cell markersΔNp63αand ABCB5,while ABCG2,ABCB5,ΔNp63 and p63αwere detectable by droplet digital PCR up to D14.Double immunolabelling revealed absence of ABCB5 staining in migrated cells but positive staining of alpha smooth muscle actin(myofibroblast marker)in some migrated cells.Droplet digital PCR showed similar expression patterns of differentiation markers but a reduction in stem cell markers between normal and keratoconic tissue with an increase in stromal cell markers and a reduction in epithelial cell markers,indicating an appropriate response to repopulating diseased tissue.CONCLUSION Cells from implanted stem cell-enriched spheres can repopulate a keratoconic corneal stromal surface in a directed manner and exhibit migratory stromal cell phenotypes.展开更多
Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons pro- mote the proliferation and oste...Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons pro- mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons （sensory neurons） from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteo- genic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera- tion of bone marrow mesenchymal stem cell-derived osteoblasts at B and 5 days of co-culture, as observed by fluorescence microscopy. The levels of mRNAs for osteogenic differentiation-re- lated factors （including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2） in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro- vides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone.展开更多
文摘Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferative regeneration. However, substantial proliferation occurs in organotypic cultures of cochleae from postnatal mice. In the present study, we studied the time course of proliferative growth in cultures of mouse cochlea explants obtained from up to 12 postnatal days. The mitotic nature of this growth was confirmed by bromodeoxyuridine (BrdU) staining and expression of proliferation cell nuclear antigen (PCNA) evaluated with real-time quantitative poly-merase chain reaction(RT-PCR). Similar growth time course was found in the cochlear explants of different postnatal ages. The new growth reached its maximum at around 2 days in culture followed by a slow-down, and virtually stopped after 5 days of culture. The possible mechanisms and the significance of this proliferation are discussed.
基金Supported by Save Sight Society of New Zealand,No.37116543New Zealand Wound Care Society,No.3713325John Hamel MacGregor Trust
文摘BACKGROUND Keratoconus is a degenerative corneal disease characterised by aberrant cell behaviour and loss of matrix that can result in vision loss.Cells extracted from peripheral corneas can form stem cell-enriched spheres,which have shown the potential to repopulate the normal peripheral corneal stroma in vitro upon sphere implantation but have not been previously studied in keratoconic tissue.AIM To investigate the therapeutic potential of stem cell-enriched spheres formed from extracted peripheral human corneal cells when introduced to keratoconic tissue.METHODS Stem cell-enriched spheres were formed from extracts of normal cadaveric human peripheral corneal cells.These spheres were implanted into incisions created in full thickness and onto the surface of 10μm thin sections of keratoconic and normal stromal tissues in vitro.Tissue sections were used to maximise use of limited keratoconic tissue available for research.Living cells were stained with Calcein-AM and visualised with stereo and fluorescence microscopy to assess survival and behaviours between the time of implantation day 0 and 14 d(D14)from implantation.Sphere cells in implanted tissues were characterised for stem cell and differentiation markers using immunohistochemistry and droplet digital PCR to assess the potential implications of these characteristics in the use of spheres in keratoconus treatment.RESULTS Spheres were successfully implanted into full-thickness central corneal tissue and onto the surface of 10μm thin en face tissue sections.No observable differences were seen in sphere migration,proliferation or differentiation in keratoconic tissue compared to normal between day 0 and D14.Spheres stained positively with Calcein-AM up to D14.Cell migration increased from day 0 to D14,occurring radially in three dimensions from the sphere and in alignment with tissue edges.Cell proliferation marker,EdU,was detected at day 10.Implanted spheres stained positively for putative stem cell markersΔNp63αand ABCB5,while ABCG2,ABCB5,ΔNp63 and p63αwere detectable by droplet digital PCR up to D14.Double immunolabelling revealed absence of ABCB5 staining in migrated cells but positive staining of alpha smooth muscle actin(myofibroblast marker)in some migrated cells.Droplet digital PCR showed similar expression patterns of differentiation markers but a reduction in stem cell markers between normal and keratoconic tissue with an increase in stromal cell markers and a reduction in epithelial cell markers,indicating an appropriate response to repopulating diseased tissue.CONCLUSION Cells from implanted stem cell-enriched spheres can repopulate a keratoconic corneal stromal surface in a directed manner and exhibit migratory stromal cell phenotypes.
基金supported by grants from the National Program on Key Basic Research Project of China(973 Program),No.2014CB542200the National Natural Science Foundation of China,No.31271284,81301570+2 种基金Program for New Century Excellent Talents in University of Ministry of Education of China,No.BMU20110270the Natural Science Foundation of Shandong Province of China,No.Y2008C18Yantai Science and Technology Development Program of China,No.2011207,2011209
文摘Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons pro- mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons （sensory neurons） from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteo- genic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera- tion of bone marrow mesenchymal stem cell-derived osteoblasts at B and 5 days of co-culture, as observed by fluorescence microscopy. The levels of mRNAs for osteogenic differentiation-re- lated factors （including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2） in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro- vides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone.
