| 1. |
- Blixt Wojciechowski, Anita, et al.
(författare)
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Long-term survival and glial differentiation of the brain-derived precursor cell line RN33B after subretinal transplantation to adult normal rats
- 2002
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 20:2, s. 163-173
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Tidskriftsartikel (refereegranskat)abstract
- The potential use of in vitro-expanded precursor cells or cell lines in repair includes transplantation of such cells for cell replacement purposes and the activation of host cells to provide "self-repair." Recently, we have reported that cells from the brain-derived cell line RN33B (derived from the embryonic rat medullary raphe and immortalized through retroviral transduction of the temperature-sensitive mutant of the simian virus 40 ([SV40] large T-antigen) survive for at least 4 weeks, integrate, and differentiate after subretinal grafting to normal adult rats. Here, we demonstrate that grafts of these cells survive for at least 4 months after subretinal transplantation to adult, normal immunosuppressed rats. Implanted cells integrate into the retinal pigment epithelium and the inner retinal layers, and the anterior part of the optic nerve. In addition, the RN33B cells migrate within the retina, occupying the whole retina from one eccentricity to the other. A large fraction of the grafted cells differentiate into glial cells, as shown by double labeling of the reporter genes LacZ or green fluorescent protein, and several glial markers, including oligodendrocytes. However, the cells did not differentiate into retinal neurons, judging from their lack of expression of retinal neuronal phenotypic markers. A significant number of the implanted cells in the host retina were in a proliferative stage, judging from proliferative cell nuclear antigen and SV40 large T-antigen immunohistochemistry. To conclude, the cells survived, integrated, and migrated over long distances within the host. Therefore, our results may be advantageous for future design of therapeutic strategies, since such cells may have the potential of being a source of, for example, growth factor delivery in experimental models of retinal degeneration.
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| 2. |
- Blixt Wojciechowski, Anita, et al.
(författare)
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Migratory capacity of the cell line RN33B and the host glial cell response after subretinal transplantation to normal adult rats
- 2004
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Ingår i: Glia. - : John Wiley & Sons. - 0894-1491 .- 1098-1136. ; 47:1, s. 58-67
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Tidskriftsartikel (refereegranskat)abstract
- As previously reported, the brain-derived precursor cell line RN33B has a great capacity to migrate when transplanted to adult brain or retina. This cell line is immortalized with the SV40 large T-antigen and carries the reporter gene LacZ and the green fluorescent protein GFP. In the present study, the precursor cells were transplanted to the subretinal space of adult rats and investigated early after grafting. The purpose was to demonstrate the migration of the grafted cells from the subretinal space into the retina and the glial cell response of the host retina. Detachment caused by the transplantation method was persistent up to 4 days after transplantation, and then reattachment occurred. The grafted cells were shown to migrate in between the photoreceptor cells before entering into the plexiform layers. Molecules involved in migration of immature neuronal cells as the polysialylated neural cell adhesion molecule (PSA-NCAM) and the collapsing response-mediated protein 4 (TUC-4) was found in the plexiform layers of the host retina, but not in the grafted cells. The expression of the intermediate filaments GFAP, vimentin, and nestin was intensely upregulated immediately after transplantation. A less pronounced upregulation was observed on sham-operated animals. In summary, the RN33B cell line migrated promptly posttransplantation and settled preferably into the plexiform layers of the retina, the same layers where the migration cues PSA-NCAM and TUC-4 were established. In addition, both the transplantation method per se and the implanted cells caused an intense glial cell response by the host retina.
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| 3. |
- Blixt Wojciechowski, Anita, et al.
(författare)
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Subretinal Transplantation of Brain-derived Precursor Cells to Young RCS Rats Promotes Photoreceptor Cell Survival☆
- 2002
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Ingår i: Experimental Eye Research. - : Elsevier. - 0014-4835 .- 1096-0007. ; 75:1, s. 23-37
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Tidskriftsartikel (refereegranskat)abstract
- The potential use of in vitro-expanded precursor cells or cell lines in brain repair includes transplantation of such cells for cell replacement purposes and the activation of host cells to provide 'self-repair'. Recently, it has been reported that the immortalized brain-derived cell line RN33B (derived from the embryonic rat medullary raphe) survive, integrate and differentiate after subretinal grafting to normal adult rats. Here, it is demonstrated that grafts of these cells survive for at least 6 weeks after implantation into postnatal days 21 and 35 retinas of normal and Royal College of Surgeons rats, a model of retinal degeneration. Implanted cells integrate into the retinal pigment epithelium and the inner retinal layers, and the anterior part of the optic nerve of both normal and Royal College of Surgeons rats. The RN33B cells migrate within the retina, occupying the whole retina from one eccentricity to the other. A significant number of the grafted cells differentiate into glial cells, as shown by the double labelling of the reporter genes LacZ or green fluorescent protein, with several glial markers, including oligodendrocytic markers. Many implanted cells in the host retina were in a proliferative stage judging from proliferative cell nuclear antigen and SV40 large T-antigen immunohistochemistry. Interestingly, there was a promotion of photoreceptor survival, extending over more than 2/3 of the superior hemisphere, in Royal College of Surgeons rats transplanted at postnatal day 21, but not at postnatal day 35. In addition, grafted cells were found in the surviving photoreceptor layer in these rats.
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| 4. |
- Blixt Wojciechowski, Anita, et al.
(författare)
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Survival and long distance migration of brain-derived precursor cells transplanted to adult rat retina
- 2004
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Ingår i: Stem Cells. - : AlphaMed Press. - 1549-4918 .- 1066-5099. ; 22:1, s. 27-38
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Tidskriftsartikel (refereegranskat)abstract
- Neural precursor cells transplanted to adult retina can integrate into the host. This is especially true when the neural precursor rat cell line RN33B is used. This cell line carries the reporter genes LacZ and green fluorescent protein (GFP). In grafted rat eyes, RN33B cells are localized from one eccentricity to the other of the host retina. In the present study, whole-mounted retinas were analyzed to obtain a more appropriate evaluation of the amount of transgene-expressing cells and the migratory capacity of these cells 3 and 8 weeks post-transplantation. Quantification was made of the number of beta-galactosidase- and GFP-expressing cells with a semiautomatized stereological cell counting system. With the same system, delineation of the distribution area of the grafted cells was also performed. At 3 weeks, 68% of the grafted eyes contained marker-expressing cells, whereas at 8 weeks only 35% of the eyes contained such cells. Counting of marker-expressing cells demonstrated a lower number of transgene-expressing cells at 3 weeks compared with 8 weeks post-transplantation. The distribution pattern of marker gene-expressing cells revealed cells occupying up to 21% at 3 weeks and up to 68% at 8 weeks of the entire host retina post-grafting. The precursor cells survived well in the adult retina although the most striking feature of the RN33B cell line was its extraordinary migratory capacity. This capability could be useful if precursor cells are used to deliver necessary genes or gene products that need to be distributed over a large diseased area.
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| 5. |
- Englund Johansson, Ulrica
(författare)
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In vivo properties of neural stem cells after transplantation into the rat brain-Studies of phenotypic differentiation and functional integration using cell-specific labelling and electrophysiological techniques
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the present thesis, we have examined the in vivo properties of in vitro expanded human and rat neural stem-and progenitor cells after transplantation into the neonatal and adult rat brain. The survival and differentiation of the grafted cells were assessed using species-specific antisera, and pre-labelling with the reporter gene green fluorescent protein (GFP). Long-term growth factor-expanded human progenitors successfully survived after grafting into the neonatal and adult striatum, subventricular zone (SVZ) and hippocampus. Target-directed migration, and region-specific neuronal differentiation of grafted cells were observed after transplantation into the neurogenic SVZ and hippocampus. Extensive migration of implanted cells identified as glial progenitors, occured within white matter. In the striatum and hippocampus, neuronal and glial differentiation were most pronounced at the graft core, with both neuronal and glial processes extending over long distances. A fraction of non-migratory undifferentiated cells remained at the implantation site. Neurogenic properties of the neural cell line RN33B, carrying the GFP reporter gene, were studied after grafting to the neonatal brain. Large numbers of RN33B cells differentiated into pyramidal neurons in the cortex and hippocampus, with projections to normal target regions, such as the thalamus and contralateral hippocampus, respectively, as revealed by retrograde tracing. Whole-cell patch clamp recordings of grafted cortical pyramidal neurons showed that RN33B cells develop physiological properties of mature neurons and become functionally integrated within host neural circuitry. Our data demonstrate a remarkable capacity of expandable neural precursors for different types of migration, and multipotential differentiation, along neuronal and glial lineages. Along with region-specific neuronal differentiation we observed establishment of appropriate anatomical projections, and functional integration into host circuitry. These results suggest that these cell types are highly useful for further research into the mechanisms responsible for cellular migration, differentiation and integration in the mature central nervous system.
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| 6. |
- Englund Johansson, Ulrica, et al.
(författare)
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Transplantation of human neural progenitor cells into the neonatal rat brain: extensive migration and differentiation with long-distance axonal projections.
- 2002
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Ingår i: Experimental Neurology. - : Elsevier BV. - 0014-4886 .- 1090-2430. ; 173:1, s. 1-21
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Tidskriftsartikel (refereegranskat)abstract
- Here we examined the ability of human neural progenitors from the embryonic forebrain, expanded for up to a year in culture in the presence of growth factors, to respond to environmental signals provided by the developing rat brain. After survival times of up to more than a year after transplantation into the striatum, the hippocampus, and the subventricular zone, the cells were analyzed using human-specific antisera and the reporter gene green fluorescent protein (GFP). From grafts implanted in the striatum, the cells migrated extensively, especially within white matter structures. Neuronal differentiation was most pronounced at the striatal graft core, with axonal projections extending caudally along the internal capsule into mesencephalon. In the hippocampus, cells migrated throughout the entire hippocampal formation and into adjacent white matter tracts, with differentiation into neurons both in the dentate gyrus and in the CA1-3 regions. Directed migration along the rostral migratory stream to the olfactory bulb and differentiation into granule cells were observed after implantation into the subventricular zone. Glial differentiation occurred at all three graft sites, predominantly at the injection sites, but also among the migrating cells. A lentiviral vector was used to transduce the cells with the GFP gene prior to grafting. The reporter gene was expressed for at least 15 weeks and the distribution of the gene product throughout the entire cytoplasmic compartment of the expressing cells allowed for a detailed morphological analysis of a portion of the grafted cells. The extensive integration and differentiation of in vitro-expanded human neural progenitor cells indicate that multipotent progenitors are capable of responding in a regionally specific manner to cues present in the developing rat brain.
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| 7. |
- Lundberg, Cecilia, et al.
(författare)
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Differentiation of the RN33B Cell Line into Forebrain Projection Neurons after Transplantation into the Neonatal Rat Brain.
- 2002
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Ingår i: Experimental Neurology. - : Elsevier BV. - 0014-4886 .- 1090-2430. ; 175:2, s. 370-387
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Tidskriftsartikel (refereegranskat)abstract
- The rat neural cell line RN33B has a remarkable ability to undergo region-specific neuronal differentiation after transplantation into the CNS. To further study its neurogenic properties in vivo, we used a recombinant lentiviral vector to genetically label the cells with the Green Fluorescent Protein (GFP) gene before implantation into the striatum/cortex, hippocampus, or mesencephalon of newborn rats. Three weeks after implantation, about 1-2% of the GFP-expressing cells had developed morphologies typical of neurons, astrocytes, or oligodendrocytes, the rest remained as either immature or undifferentiated nestin-positive cells. At 15-17 weeks postgrafting, the immature cells had disappeared in most graft recipients and only cells with neuronal or glial morphologies remained in similar numbers as at 3 weeks. The GFP distributed throughout the expressing cells, revealing fine morphological details, including dendrites with spines and extensive axonal projections. In all forebrain regions, the grafted cells differentiated into neurons with morphologies characteristic for each site, including large numbers of pyramidal-like cells in the cortex and the hippocampus, giving rise to dense projections to normal cortical target regions and to the contralateral hippocampus, respectively. In lower numbers, it was also possible to identify GFP-positive granulelike cells in the hippocampus, as well as densely spiny neurons in the striatum. In the mesencephalon by contrast, cells with astrocytic features predominated. The ability of the grafted RN33B cells to undergo region-specific differentiation into highly specialized types of forebrain projection neurons and establish connections with appropriate targets suggests that cues present in the microenvironment of the neonatal rat brain can effectively guide the development of immature progenitors, also in the absence of ongoing neurogenesis. (c) 2002 Elsevier Science (USA).
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| 8. |
- Warfvinge, Karin, et al.
(författare)
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Retinal Integration of Grafts of Brain-Derived Precursor Cell Lines Implanted Subretinally into Adult, Normal Rats
- 2001
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Ingår i: Experimental Neurology. - : Elsevier. - 0014-4886 .- 1090-2430. ; 169:1, s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- The ability of in vitro-expanded neural precursor cells or cell lines to differentiate following transplantation has significant implications for current research on central nervous system repair. Recently, interest has been focussed on grafts of such neural precursors implanted also into the eye or retina. Here, we demonstrate with a non-traumatizing subretinal transplantation method, that grafts of the two immortalized brain-derived cell lines C 17-2 (from postnatal mouse cerebellum) and RN33B (from the embryonic rat medullary raphe) survive for at least up to four weeks, after implantation into the adult normal rat retina. For both cell lines, implanted cells gradually integrate into all major retinal cell layers, including the retinal pigment epithelium, and judged by the morphology differentiate into both glial- and neuron-like cells, as shown by thymidine autoradiography, mouse-specific in situ hybridization, and using immunohistochemistry to detect the reporter gene LacZ. Our results suggest that these and other similar neural cell lines could be very useful in the continuos experiments in models of retinal disorders to further assess both the cell replacement and ex vivo gene therapy approaches.
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