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Grafting and early expression of growth factors from adipose-derived stem cells transplanted into the cochlea, in a Guinea pig model of acoustic trauma.

Fetoni AR, Lattanzi W, Eramo SL, Barba M, Paciello F, Moriconi C, Rolesi R, Michetti F, Troiani D, Paludetti G - Front Cell Neurosci (2014)

Bottom Line: ASC implantation did not modify auditory function.ASCs migrated from the perilymphatic to the endolymphatic compartment, during the analyzed time course.Immunofluorescence confirmed the increased expression, which appeared to be further strengthened by ASCs' implantation.

View Article: PubMed Central - PubMed

Affiliation: Department of Head and Neck Surgery, Università Cattolica del Sacro Cuore , Rome , Italy.

ABSTRACT
Noise exposure causes damage of multiple cochlear cell types producing permanent hearing loss with important social consequences. In mammals, no regeneration of either damaged hair cells or auditory neurons has been observed and no successful treatment is available to achieve a functional recovery. Loads of evidence indicate adipose-derived stem cells (ASCs) as promising tools in diversified regenerative medicine applications, due to the high degree of plasticity and trophic features. This study was aimed at identifying the path of in vivo cell migration and expression of trophic growth factors, upon ASCs transplantation into the cochlea, following noise-induced injury. ASCs were isolated in primary culture from the adipose tissue of a guinea pig, transduced using a viral vector to express the green fluorescent protein, and implanted into the scala tympani of deafened animals. Auditory function was assessed 3 and 7 days after surgery. The expression of trophic growth factors was comparatively analyzed using real-time PCR in control and noise-injured cochlear tissues. Immunofluorescence was used to assess the in vivo localization and expression of trophic growth factors in ASCs and cochleae, 3 and 7 days following homologous implantation. ASC implantation did not modify auditory function. ASCs migrated from the perilymphatic to the endolymphatic compartment, during the analyzed time course. Upon noise exposure, the expression of chemokine ligands and receptors related to the PDGF, VEGF, and TGFbeta pathways, increased in the cochlear tissues, possibly guiding in vivo cell migration. Immunofluorescence confirmed the increased expression, which appeared to be further strengthened by ASCs' implantation. These results indicated that ASCs are able to migrate at the site of tissue damage and express trophic factors, upon intracochlear implantation, providing an original proof of principle, which could pave the way for further developments of ASC-based treatments of deafness.

No MeSH data available.


Related in: MedlinePlus

TGFβ and PDGFR expression in the cochlea. Representative images from confocal microscopy analysis of cochlear cryosections collected at day 7 after surgery. TGFβ (red) and DAPI nuclear staining (blue) in (A) control unexposed cochleae; (B) GFP-positive cells are located close to the Reissner’s membrane (arrow) in the scala media (the faint green fluorescence shown in cochlear structures was observed in control negative sections due to a slight spontaneous fluorescence, data not shown); (C) in the noise + vehicle group (TGFβ fluorescence observed in the spiral ganglion, organ of Corti and stria vascularis); (D) after ASC implantation, red fluorescence is observed in the scala media and in implanted ASCs near the lateral wall (stria vascularis/arrow head and spiral ligament), in the spiral ganglion (asterisk), and Reissner’s membrane (arrow), as shown at higher magnification (E–G), where ASCs attached to the Reissner’s membrane are shown. In lower panels, PDGFR staining is shown in unexposed (H) and noise-exposed cochleae [(J) red fluorescence in the organ of Corti, spiral ganglion, and stria vascularis]; (I) green fluorescent ASCs in the noise-exposed cochlea; (K) PDGFR increased fluorescence in noise + ASCs group in the stria vascularis (arrow head) and in the spiral ganglion (asterisk). TGFβ, transforming growth factor β; PDGFR, platelet-derived growth factor receptor.
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Figure 5: TGFβ and PDGFR expression in the cochlea. Representative images from confocal microscopy analysis of cochlear cryosections collected at day 7 after surgery. TGFβ (red) and DAPI nuclear staining (blue) in (A) control unexposed cochleae; (B) GFP-positive cells are located close to the Reissner’s membrane (arrow) in the scala media (the faint green fluorescence shown in cochlear structures was observed in control negative sections due to a slight spontaneous fluorescence, data not shown); (C) in the noise + vehicle group (TGFβ fluorescence observed in the spiral ganglion, organ of Corti and stria vascularis); (D) after ASC implantation, red fluorescence is observed in the scala media and in implanted ASCs near the lateral wall (stria vascularis/arrow head and spiral ligament), in the spiral ganglion (asterisk), and Reissner’s membrane (arrow), as shown at higher magnification (E–G), where ASCs attached to the Reissner’s membrane are shown. In lower panels, PDGFR staining is shown in unexposed (H) and noise-exposed cochleae [(J) red fluorescence in the organ of Corti, spiral ganglion, and stria vascularis]; (I) green fluorescent ASCs in the noise-exposed cochlea; (K) PDGFR increased fluorescence in noise + ASCs group in the stria vascularis (arrow head) and in the spiral ganglion (asterisk). TGFβ, transforming growth factor β; PDGFR, platelet-derived growth factor receptor.

Mentions: Morphological analysis of cochlear sections revealed no signs of relevant traumatic damage to the basilar membrane, the organ of Corti, and the Reissner’s membrane, attributable to the surgical procedure, in any of the tested animals. ASCs were clearly distinguished from the endogenous cells based on their transgenic GFP expression, which co-localized with DAPI-stained nuclei (Figures 4A–C), and tended to aggregate close to each other, as observed in cochlear specimens 3 and 7 days after implantation (Figures 4D–G) and they were thus recognized as exogenous cells into the labyrinthine fluid compartments. Namely, at day 3, in the Sham + ASCs specimens, the transplanted cells were found as a cluster of green fluorescent ASCs in the perilymphatic spaces mainly attached to the walls of the scala tympani (Figure 4D); however, in the noise-exposed + ASCs ears, some ACSs migrated to the scala vestibuli (Figure 4E); few cells were also found attached to the basilar membrane in the scala tympani and in the proximity of Rosenthal canal (Figures 4D,E). At day 7, in the Sham + ASCs ears, the green fluorescent cells in the perilymphatic spaces of the scala tympani decreased, while they increased in the scala vestibuli (Figure 4F). Interestingly, in the noise-exposed + ASCs ears, the fluorescence was also found in the scala media close to the tectorial membrane and the stria vascularis indicating that ASCs migrated in the scala vestibuli and in the scala media (Figure 4G). Some ASCs were detected nearby the Reissner’s membrane (see also Figures 5 and 6).


Grafting and early expression of growth factors from adipose-derived stem cells transplanted into the cochlea, in a Guinea pig model of acoustic trauma.

Fetoni AR, Lattanzi W, Eramo SL, Barba M, Paciello F, Moriconi C, Rolesi R, Michetti F, Troiani D, Paludetti G - Front Cell Neurosci (2014)

TGFβ and PDGFR expression in the cochlea. Representative images from confocal microscopy analysis of cochlear cryosections collected at day 7 after surgery. TGFβ (red) and DAPI nuclear staining (blue) in (A) control unexposed cochleae; (B) GFP-positive cells are located close to the Reissner’s membrane (arrow) in the scala media (the faint green fluorescence shown in cochlear structures was observed in control negative sections due to a slight spontaneous fluorescence, data not shown); (C) in the noise + vehicle group (TGFβ fluorescence observed in the spiral ganglion, organ of Corti and stria vascularis); (D) after ASC implantation, red fluorescence is observed in the scala media and in implanted ASCs near the lateral wall (stria vascularis/arrow head and spiral ligament), in the spiral ganglion (asterisk), and Reissner’s membrane (arrow), as shown at higher magnification (E–G), where ASCs attached to the Reissner’s membrane are shown. In lower panels, PDGFR staining is shown in unexposed (H) and noise-exposed cochleae [(J) red fluorescence in the organ of Corti, spiral ganglion, and stria vascularis]; (I) green fluorescent ASCs in the noise-exposed cochlea; (K) PDGFR increased fluorescence in noise + ASCs group in the stria vascularis (arrow head) and in the spiral ganglion (asterisk). TGFβ, transforming growth factor β; PDGFR, platelet-derived growth factor receptor.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4202717&req=5

Figure 5: TGFβ and PDGFR expression in the cochlea. Representative images from confocal microscopy analysis of cochlear cryosections collected at day 7 after surgery. TGFβ (red) and DAPI nuclear staining (blue) in (A) control unexposed cochleae; (B) GFP-positive cells are located close to the Reissner’s membrane (arrow) in the scala media (the faint green fluorescence shown in cochlear structures was observed in control negative sections due to a slight spontaneous fluorescence, data not shown); (C) in the noise + vehicle group (TGFβ fluorescence observed in the spiral ganglion, organ of Corti and stria vascularis); (D) after ASC implantation, red fluorescence is observed in the scala media and in implanted ASCs near the lateral wall (stria vascularis/arrow head and spiral ligament), in the spiral ganglion (asterisk), and Reissner’s membrane (arrow), as shown at higher magnification (E–G), where ASCs attached to the Reissner’s membrane are shown. In lower panels, PDGFR staining is shown in unexposed (H) and noise-exposed cochleae [(J) red fluorescence in the organ of Corti, spiral ganglion, and stria vascularis]; (I) green fluorescent ASCs in the noise-exposed cochlea; (K) PDGFR increased fluorescence in noise + ASCs group in the stria vascularis (arrow head) and in the spiral ganglion (asterisk). TGFβ, transforming growth factor β; PDGFR, platelet-derived growth factor receptor.
Mentions: Morphological analysis of cochlear sections revealed no signs of relevant traumatic damage to the basilar membrane, the organ of Corti, and the Reissner’s membrane, attributable to the surgical procedure, in any of the tested animals. ASCs were clearly distinguished from the endogenous cells based on their transgenic GFP expression, which co-localized with DAPI-stained nuclei (Figures 4A–C), and tended to aggregate close to each other, as observed in cochlear specimens 3 and 7 days after implantation (Figures 4D–G) and they were thus recognized as exogenous cells into the labyrinthine fluid compartments. Namely, at day 3, in the Sham + ASCs specimens, the transplanted cells were found as a cluster of green fluorescent ASCs in the perilymphatic spaces mainly attached to the walls of the scala tympani (Figure 4D); however, in the noise-exposed + ASCs ears, some ACSs migrated to the scala vestibuli (Figure 4E); few cells were also found attached to the basilar membrane in the scala tympani and in the proximity of Rosenthal canal (Figures 4D,E). At day 7, in the Sham + ASCs ears, the green fluorescent cells in the perilymphatic spaces of the scala tympani decreased, while they increased in the scala vestibuli (Figure 4F). Interestingly, in the noise-exposed + ASCs ears, the fluorescence was also found in the scala media close to the tectorial membrane and the stria vascularis indicating that ASCs migrated in the scala vestibuli and in the scala media (Figure 4G). Some ASCs were detected nearby the Reissner’s membrane (see also Figures 5 and 6).

Bottom Line: ASC implantation did not modify auditory function.ASCs migrated from the perilymphatic to the endolymphatic compartment, during the analyzed time course.Immunofluorescence confirmed the increased expression, which appeared to be further strengthened by ASCs' implantation.

View Article: PubMed Central - PubMed

Affiliation: Department of Head and Neck Surgery, Università Cattolica del Sacro Cuore , Rome , Italy.

ABSTRACT
Noise exposure causes damage of multiple cochlear cell types producing permanent hearing loss with important social consequences. In mammals, no regeneration of either damaged hair cells or auditory neurons has been observed and no successful treatment is available to achieve a functional recovery. Loads of evidence indicate adipose-derived stem cells (ASCs) as promising tools in diversified regenerative medicine applications, due to the high degree of plasticity and trophic features. This study was aimed at identifying the path of in vivo cell migration and expression of trophic growth factors, upon ASCs transplantation into the cochlea, following noise-induced injury. ASCs were isolated in primary culture from the adipose tissue of a guinea pig, transduced using a viral vector to express the green fluorescent protein, and implanted into the scala tympani of deafened animals. Auditory function was assessed 3 and 7 days after surgery. The expression of trophic growth factors was comparatively analyzed using real-time PCR in control and noise-injured cochlear tissues. Immunofluorescence was used to assess the in vivo localization and expression of trophic growth factors in ASCs and cochleae, 3 and 7 days following homologous implantation. ASC implantation did not modify auditory function. ASCs migrated from the perilymphatic to the endolymphatic compartment, during the analyzed time course. Upon noise exposure, the expression of chemokine ligands and receptors related to the PDGF, VEGF, and TGFbeta pathways, increased in the cochlear tissues, possibly guiding in vivo cell migration. Immunofluorescence confirmed the increased expression, which appeared to be further strengthened by ASCs' implantation. These results indicated that ASCs are able to migrate at the site of tissue damage and express trophic factors, upon intracochlear implantation, providing an original proof of principle, which could pave the way for further developments of ASC-based treatments of deafness.

No MeSH data available.


Related in: MedlinePlus