| 1. |
- Glavaski-Joksimovic, A., et al.
(författare)
-
Morphological differentiation of tau–green fluorescent protein embryonic stem cells into neurons after co-culture with auditory brain stem slices
- 2009
-
Ingår i: Neuroscience. - : Elsevier. - 0306-4522 .- 1873-7544. ; 162:2, s. 472-481
-
Tidskriftsartikel (refereegranskat)abstract
- Most types of congenital and acquired hearing loss are caused by loss of sensory hair cells in the inner ear and their respective afferent neurons. Replacement of spiral ganglion neurons (SGN) would therefore be one prioritized step in an attempt to restore sensory neuronal hearing loss. To initiate an SGN repair paradigm we previously transplanted embryonic neuronal tissue and stem cells (SC) into the inner ear in vivo. The results illustrated good survival of the implant. One such repair, however, would not have any clinical significance unless central connections from the implanted SIGN could be established. For the purpose of evaluating the effects of cell transplantation on cochlear nucleus (CN) neurons we have established organotypic brain stem (BS) cultures containing the CN. At present we have used in vitro techniques to study the survival and differentiation of tau-green fluorescent protein (GFP) mouse embryonic stem cells (MESC) as a mono- or co-culture with BS slices. For the co-culture, 300 mu m thick auditory BS slices encompassing the CN were prepared from postnatal Sprague-Dawley rats. The slices were propagated using the membrane interface method and the CN neurons labeled with Dil. After 5 +/- 2 days in culture a tau-GFP MESC suspension was deposited next to CN in the BS slice. Following deposition the MESC migrated towards the CN. One and two weeks after transplantation the co-cultures were fixed and immunostained with antibodies raised against neuroprogenitor, neuronal, glial and synaptic vesicle protein markers. Our experiments with the tau-GFP MESC and auditory BS co-cultures show a significant MESC survival but also differentiation into neuronal cells. The findings illustrate the significance of SC and auditory BS co-cultures regarding survival, migration, neuronal differentiation and connections.
|
|
| 2. |
- Glavaski-Joksimovic, Aleksandra, et al.
(författare)
-
Survival, migration, and differentiation of Sox1-GFP embryonic stem cells in coculture with an auditory brainstem slice preparation
- 2008
-
Ingår i: Cloning and Stem Cells. - : Mary Ann Liebert. - 1536-2302 .- 1557-7457. ; 10:1, s. 75-87
-
Tidskriftsartikel (refereegranskat)abstract
- The poor regeneration capability of the mammalian hearing organ has initiated different approaches to enhance its functionality after injury. To evaluate a potential neuronal repair paradigm in the inner ear and cochlear nerve we have previously used embryonic neuronal tissue and stem cells for implantation in vivo and in vitro. At present, we have used in vitro techniques to study the survival and differentiation of Sox1-green fluorescent protein (GFP) mouse embryonic stem (ES) cells as a monoculture or as a coculture with rat auditory brainstem slices. For the coculture, 300 mu m-thick brainstem slices encompassing the cochlear nucleus and cochlear nerve were prepared from postnatal SD rats. The. slices were propagated using the membrane interface method and the cochlear nuclei were prelabeled with DiI. After some days in culture a suspension of Sox1 cells was deposited next to the brainstem slice. Following. deposition Sox1 cells migrated toward the brainstem and onto the cochlear nucleus. GFP was not detectable in undifferentiated ES cells but became evident during neural differentiation.. Up to 2 weeks after transplantation the cocultures were fixed. The undifferentiated cells were evaluated with antibodies against progenitor cells whereas the differentiated cells were determined with neuronal and glial markers. The morphological and immunohistochemical data indicated that Sox1 cells in monoculture differentiated into a higher percentage of glial cells than neurons. However, when a coculture was used a significantly lower percentage of Sox1 cells differentiated into glial cells. The results demonstrate that a coculture of Sox1 cells and auditory brainstem present a useful model to study stem cell differentiation.
|
|
| 3. |
- Herlenius, E., et al.
(författare)
-
Functional stem cell integration assessed by organotypic slice cultures
- 2012
-
Ingår i: Current Protocols in Stem Cell Biology. - : John Wiley & Sons. - 1941-7322 .- 1938-8969. ; 1:SUPPL.23
-
Tidskriftsartikel (refereegranskat)abstract
- Re-formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell-mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host. One option for in vitro studies is to examine the integration of exogenous stem cells into an existing active neuronal network in ex vivo organotypic cultures. Organotypic cultures leave the structural integrity essentially intact while still allowing the microenvironment to be carefully controlled. This allows detailed studies over time of cellular responses and cellcell interactions, which are not readily performed in vivo. This unit describes procedures for using organotypic slice cultures as ex vivo model systems for studying neural stem cell and embryonic stem cell engraftment and communication with CNS host tissue.
|
|
| 4. |
- Hertegard, S., et al.
(författare)
-
Viscoelastic and histologic properties in scarred rabbit vocal folds after mesenchymal stem cell injection
- 2006
-
Ingår i: The Laryngoscope. - : Wiley-Blackwell. - 0023-852X .- 1531-4995. ; 116:7, s. 1248-1254
-
Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE/HYPOTHESIS:The aim of this study was to analyze the short-term viscoelastic and histologic properties of scarred rabbit vocal folds after injection of human mesenchymal stem cells (MSC) as well as the degree of MSC survival. Because MSCs are antiinflammatory and regenerate mesenchymal tissues, can MSC injection reduce vocal fold scarring after injury?STUDY DESIGN:Twelve vocal folds from 10 New Zealand rabbits were scarred by a localized resection and injected with human MSC or saline. Eight vocal folds were left as controls.MATERIAL AND METHODS:After 4 weeks, 10 larynges were stained for histology and evaluation of the lamina propria thickness. Collagen type I content was analyzed from six rabbits. MSC survival was analyzed by fluorescent in situ hybridization staining from three rabbits. Viscoelasticity for 10 vocal folds was analyzed in a parallel-plate rheometer.RESULTS:The rheometry on fresh-frozen samples showed decreased dynamic viscosity and lower elastic modulus (P<.01) in the scarred samples injected with MSC as compared with the untreated scarred group. Normal controls had lower dynamic viscosity and elastic modulus as compared with the scarred untreated and treated vocal folds (P<.01). Histologic analysis showed a higher content of collagen type 1 in the scarred samples as compared with the normal vocal folds and with the scarred folds treated with MSC. MSCs remained in all samples analyzed.CONCLUSIONS:The treated scarred vocal folds showed persistent MSC. Injection of scarred rabbit vocal folds with MSC rendered improved viscoelastic parameters and less signs of scarring expressed as collagen content in comparison to the untreated scarred vocal folds.
|
|
| 5. |
- Hu, Zhengqing, et al.
(författare)
-
Functional Evaluation of a Cell Replacement Therapy in the Inner Ear
- 2009
-
Ingår i: Otology and Neurotology. - : Lippincott Williams & Wilkins. - 1531-7129 .- 1537-4505. ; 30:4, s. 551-558
-
Tidskriftsartikel (refereegranskat)abstract
- HYPOTHESIS:Cell replacement therapy in the inner ear will contribute to the functional recovery of hearing loss.BACKGROUND:Cell replacement therapy is a potentially powerful approach to replace degenerated or severely damaged spiral ganglion neurons. This study aimed at stimulating the neurite outgrowth of the implanted neurons and enhancing the potential therapeutic of inner ear cell implants.METHODS:Chronic electrical stimulation (CES) and exogenous neurotrophic growth factor (NGF) were applied to 46 guinea pigs transplanted with embryonic dorsal root ganglion (DRG) neurons 4 days postdeafening. The animals were evaluated with the electrically evoked auditory brainstem responses (EABRs) at experimental Days 7, 11, 17, 24, and 31. The animals were euthanized at Day 31, and the inner ears were dissected for immunohistochemistry investigation.RESULTS:Implanted DRG cells, identified by enhanced green fluorescent protein fluorescence and a neuronal marker, were found close to Rosenthal canal in the adult inner ear for up to 4 weeks after transplantation. Extensive neurite projections clearly, greater than in nontreated animals, were observed to penetrate the bony modiolus and reach the spiral ganglion region in animals supplied with CES and/or NGF. There was, however, no significant difference in the thresholds of EABRs between DRG-transplanted animals supplied with CES and/or NGF and DRG-transplanted animals without CES or NGF supplement.CONCLUSION:The results suggest that CES and/or NGF can stimulate neurite outgrowth from implanted neurons, although based on EABR measurement, these interventions did not induce functional connections to the central auditory pathway. Additional time or novel approaches may enhance functional responsiveness of implanted cells in the adult cochlea
|
|
| 6. |
- Hu, Z., et al.
(författare)
-
NGF stimulates extensive neurite outgrowth from implanted dorsal root ganglion neurons following transplantation into the adult rat inner ear
- 2005
-
Ingår i: Neurobiology of Disease. - : Elsevier. - 0969-9961 .- 1095-953X. ; 18:1, s. 184-192
-
Tidskriftsartikel (refereegranskat)abstract
- Neuronal tissue transplantation is a potential way to replace degenerated spiral ganglion neurons (SGNs) since these cells cannot regenerate in adult mammals. To investigate whether nerve growth factor (NGF) can stimulate neurite outgrowth from implanted neurons, mouse embryonic dorsal root ganglion (DRG) cells expressing enhanced green fluorescent protein (EGFP) were transplanted into the scala tympani of adult rats with a supplement of NGF or artificial perilymph. DRG neurons were observed in the cochlea for up to 6 weeks postoperatively. A significant difference was identified in the number of DRG neurons between the NGF and non-NGF groups. In the NGF group, extensive neurite projections from DRGs were found penetrating the osseous modiolus towards the spiral ganglion. These results suggest the possibility that embryonic neuronal implants may become integrated within the adult auditory nervous system. In combination with a cochlear prosthesis, a neuronal implantation strategy may provide a possibility for further treatment of profoundly deaf patients.
|
|
| 7. |
- Jiao, Yu, et al.
(författare)
-
BDNF Increases Survival and Neuronal Differentiation of Human Neural Precursor Cells Cotransplanted with a Nanofiber Gel to the Auditory Nerve in a Rat Model of Neuronal Damage
- 2014
-
Ingår i: BioMed Research International. - : Hindawi Publishing Corporation. - 2314-6133 .- 2314-6141. ; 2014
-
Tidskriftsartikel (refereegranskat)abstract
- Objectives. To study possible nerve regeneration of a damaged auditory nerve by the use of stem cell transplantation. Methods. We transplanted HNPCs to the rat AN trunk by the internal auditory meatus (IAM). Furthermore, we studied if addition of BDNF affects survival and phenotypic differentiation of the grafted HNPCs. A bioactive nanofiber gel (PA gel), in selected groups mixed with BDNF, was applied close to the implanted cells. Before transplantation, all rats had been deafened by a round window niche application of β-bungarotoxin. This neurotoxin causes a selective toxic destruction of the AN while keeping the hair cells intact. Results. Overall, HNPCs survived well for up to six weeks in all groups. However, transplants receiving the BDNF-containing PA gel demonstrated significantly higher numbers of HNPCs and neuronal differentiation. At six weeks, a majority of the HNPCs had migrated into the brain stem and differentiated. Differentiated human cells as well as neurites were observed in the vicinity of the cochlear nucleus. Conclusion. Our results indicate that human neural precursor cells (HNPC) integration with host tissue benefits from additional brain derived neurotrophic factor (BDNF) treatment and that these cells appear to be good candidates for further regenerative studies on the auditory nerve (AN).
|
|
| 8. |
- Jiao, Yu, et al.
(författare)
-
Olfactory ensheathing cells promote neurite outgrowth from co-cultured brain stem slice
- 2011
-
Ingår i: EXPERIMENTAL NEUROLOGY. - : Elsevier Science B.V., Amsterdam. - 0014-4886 .- 1090-2430. ; 229:1, s. 65-71
-
Tidskriftsartikel (refereegranskat)abstract
- Cell therapy aiming at the replacement of degenerated neurons is a very attractive approach. By using an established in vitro organotypic brain stem (BS) slice culture we screen for candidate donor cells, some of them being further functionally assessed in in vivo models of sensorineural hearing loss. Both in vitro and in vivo systems show that implanted cells face challenges of survival, targeted migration, differentiation and functional integration with the host tissue. Low success rates are possibly due to the lack of necessary neurotrophic factors, adhesion molecules and guiding cues. Olfactory ensheathing cells (OECs) have been shown to express a number of neurotrophic factors and to promote axonal growth through cell to cell interactions. In the present study we co-cultured OECs with organotypic BS slice in order to see if OECs can serve as a facilitator when screening candidate donor cells in an organotypic culture setup. Here we show that OECs when co-cultured with the auditory BS slice not only promote neurite outgrowth from the cochlear nucleus (CN) region of the BS slice but also support cells by having BS slice axons growing along their processes. These findings further suggest that OECs may enhance survival and targeted migration of candidate donor cells suitable for cell therapy in vitro and in vivo. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.
|
|
| 9. |
- Kaiser, Andreas, et al.
(författare)
-
Brain stern slice conditioned medium contains endogenous BDNF and GDNF that affect neural crest boundary cap cells in co-culture
- 2014
-
Ingår i: Brain Research. - : Elsevier. - 0006-8993 .- 1872-6240. ; 1566, s. 12-23
-
Tidskriftsartikel (refereegranskat)abstract
- Conditioned medium (CM), made by collecting medium after a few days in cell culture and then re-using it to further stimulate other cells, is a known experimental concept since the 1950s. Our group has explored this technique to stimulate the performance of cells in culture in general, and to evaluate stem- and progenitor cell aptitude for auditory nerve repair enhancement in particular. As compared to other mediums, all primary endpoints in our published experimental settings have weighed in favor of conditioned culture medium, where we have shown that conditioned culture medium has a stimulatory effect on cell survival. In order to explore the reasons for this improved survival we set out to analyze the conditioned culture medium. We utilized ELISA kits to investigate whether brain stem (BS) slice CM contains any significant amounts of brain-derived neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF). We further looked for a donor cell with progenitor characteristics that would be receptive to BDNF and GDNF. We chose the well-documented boundary cap (BC) progenitor cells to be tested in our in vitro co-culture setting together with cochlear nucleus (CN) of the BS. The results show that BS CM contains BDNF and GDNF and that survival of BC cells, as well as BC cell differentiation into neurons, were enhanced when BS CM were used. Altogether, we conclude that BC cells transplanted into a BDNF and GDNF rich environment could be suitable for treatment of a traumatized or degenerated auditory nerve.
|
|
| 10. |
- Kaiser, Andreas, et al.
(författare)
-
The Effects of Matrigel® on the Survival and Differentiation of a Human Neural Progenitor Dissociated Sphere Culture
- 2020
-
Ingår i: Anatomical Record Part A-discoveries in Molecular Cellular and Evolutionary Biology. - : Wiley. - 1552-4884 .- 1932-8494 .- 1932-8486. ; 303:3, s. 441-450
-
Tidskriftsartikel (refereegranskat)abstract
- We have previously developed an in vitro organotypic culture setting in order to investigate the performance of cellular substrates transplanted to the auditory nervous system. We have utilized this system to predict the efficacy of human neural progenitor cells (HNPCs) in transplantation to the auditory nerve to facilitate regeneration of sensory auditory nerve structures in vivo and in vitro. To optimize the growth and differentiation of HNPCs we have introduced an expansion of our in vitro system, exploring the impact of a growth factor‐altered microenvironment. Here, we seeded HNPCs as a dissociated sphere culture on a hydrogel matrix coating (Matrigel®). We evaluated the performance of HNPCs by studying their survival, differentiation, and their axon‐forming capacity. In identical culture conditions, we found that the overall survival rate of HNPCs on Matrigel coated surfaces was better than that on surfaces that were not coated with Matrigel. Furthermore, cells on Matrigel differentiated into neuronal cells to a far greater extent leading to strong synaptic marker signatures. Overall, our findings show that the present Matrigel matrix setting offers an experimental environment for the HNPCs to grow where these cells show novel and promising phenotypic characteristics suitable for further in vivo transplantation to the auditory nerve.
|
|
