Limits...
A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

Herget M, Scheibinger M, Guo Z, Jan TA, Adams CM, Cheng AG, Heller S - PLoS ONE (2013)

Bottom Line: Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level.Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells.Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology - HNS, Stanford University, Stanford, California, USA.

ABSTRACT
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Show MeSH

Related in: MedlinePlus

Qualitative analysis of identified non-sensory cell markers by immunocytochemistry.Shown are cross sections of E18 chicken utricles. (A) Co-immunolabeling with antibodies to collagen XVII (green), the hair cell marker myosin VIIA (red), and Sox2 (blue). Collagen XVII immunoreactivity was detected in the basal lamina and below the basal lamina in the mesenchymal stromal cell layer. (B) Antibodies to talin detected the protein in supporting cells, which are Sox2 (blue) immunopositive and myosin VIIA (green)-negative. Talin was also detectable in mesenchymal stromal cells. (C) To better visualize the location of talin immunoreactivity in supporting cells, we used light microscopic images and the Sox2 immunostaining to outline the supporting cells’ nuclei (labeled 1–4), and found association of talin immunoreactivity (green) perinuclear and toward the basal end of supporting cells (arrows).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672136&req=5

pone-0066026-g008: Qualitative analysis of identified non-sensory cell markers by immunocytochemistry.Shown are cross sections of E18 chicken utricles. (A) Co-immunolabeling with antibodies to collagen XVII (green), the hair cell marker myosin VIIA (red), and Sox2 (blue). Collagen XVII immunoreactivity was detected in the basal lamina and below the basal lamina in the mesenchymal stromal cell layer. (B) Antibodies to talin detected the protein in supporting cells, which are Sox2 (blue) immunopositive and myosin VIIA (green)-negative. Talin was also detectable in mesenchymal stromal cells. (C) To better visualize the location of talin immunoreactivity in supporting cells, we used light microscopic images and the Sox2 immunostaining to outline the supporting cells’ nuclei (labeled 1–4), and found association of talin immunoreactivity (green) perinuclear and toward the basal end of supporting cells (arrows).

Mentions: For non-sensory cells, collagen XVIII alpha 1 and talin were identified by mass spectrometry, and monoclonal antibodies to these two chicken proteins detected them in association with non-sensory and supporting cells and not hair cells in the E18 utricle (Fig. 8). Antibodies to the extracellular matrix protein collagen XVIII [36], which was identified in the non-sensory cell only fraction (Table 2), labeled the basal lamina directly underneath the non-sensory cell layer. No immunoreactivity was detectable in hair cells. This localization, combined with the mass spectrometry data suggests that non-sensory and presumably supporting cells secrete collagen XVIII, but it cannot exclude a possible contribution of mesenchymal stromal cells to the mass spectrometry data because these cells were also labeled with collagen XVIII antibodies (Fig. 8A). The cytoskeletal protein talin, which is found concentrated at focal adhesion points and at points of cell-substratum contact [37], was identified via Fisher exact analysis as highly enriched by non-sensory cells compared to hair cells. Monoclonal antibodies detected the protein in supporting cells as well as in mesenchymal stromal cells, but not in hair cells (Fig. 8B,C).


A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

Herget M, Scheibinger M, Guo Z, Jan TA, Adams CM, Cheng AG, Heller S - PLoS ONE (2013)

Qualitative analysis of identified non-sensory cell markers by immunocytochemistry.Shown are cross sections of E18 chicken utricles. (A) Co-immunolabeling with antibodies to collagen XVII (green), the hair cell marker myosin VIIA (red), and Sox2 (blue). Collagen XVII immunoreactivity was detected in the basal lamina and below the basal lamina in the mesenchymal stromal cell layer. (B) Antibodies to talin detected the protein in supporting cells, which are Sox2 (blue) immunopositive and myosin VIIA (green)-negative. Talin was also detectable in mesenchymal stromal cells. (C) To better visualize the location of talin immunoreactivity in supporting cells, we used light microscopic images and the Sox2 immunostaining to outline the supporting cells’ nuclei (labeled 1–4), and found association of talin immunoreactivity (green) perinuclear and toward the basal end of supporting cells (arrows).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672136&req=5

pone-0066026-g008: Qualitative analysis of identified non-sensory cell markers by immunocytochemistry.Shown are cross sections of E18 chicken utricles. (A) Co-immunolabeling with antibodies to collagen XVII (green), the hair cell marker myosin VIIA (red), and Sox2 (blue). Collagen XVII immunoreactivity was detected in the basal lamina and below the basal lamina in the mesenchymal stromal cell layer. (B) Antibodies to talin detected the protein in supporting cells, which are Sox2 (blue) immunopositive and myosin VIIA (green)-negative. Talin was also detectable in mesenchymal stromal cells. (C) To better visualize the location of talin immunoreactivity in supporting cells, we used light microscopic images and the Sox2 immunostaining to outline the supporting cells’ nuclei (labeled 1–4), and found association of talin immunoreactivity (green) perinuclear and toward the basal end of supporting cells (arrows).
Mentions: For non-sensory cells, collagen XVIII alpha 1 and talin were identified by mass spectrometry, and monoclonal antibodies to these two chicken proteins detected them in association with non-sensory and supporting cells and not hair cells in the E18 utricle (Fig. 8). Antibodies to the extracellular matrix protein collagen XVIII [36], which was identified in the non-sensory cell only fraction (Table 2), labeled the basal lamina directly underneath the non-sensory cell layer. No immunoreactivity was detectable in hair cells. This localization, combined with the mass spectrometry data suggests that non-sensory and presumably supporting cells secrete collagen XVIII, but it cannot exclude a possible contribution of mesenchymal stromal cells to the mass spectrometry data because these cells were also labeled with collagen XVIII antibodies (Fig. 8A). The cytoskeletal protein talin, which is found concentrated at focal adhesion points and at points of cell-substratum contact [37], was identified via Fisher exact analysis as highly enriched by non-sensory cells compared to hair cells. Monoclonal antibodies detected the protein in supporting cells as well as in mesenchymal stromal cells, but not in hair cells (Fig. 8B,C).

Bottom Line: Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level.Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells.Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology - HNS, Stanford University, Stanford, California, USA.

ABSTRACT
Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Show MeSH
Related in: MedlinePlus