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Intrinsic regenerative potential of murine cochlear supporting cells

ABSTRACT: P3 Math1-nGFP mouse cochlear sensory epithelia, consisting of greater and lesser epithelial ridges (GER & LER) including the organ of Corti with nGFP-positive hair cells, were dissected, dissociated into single cells, and labeled with propidium iodide and three CD marker antibodies. The cell suspension was subjected to FACS purification. 83.6 ±2.8% of the total input of cells were viable, determined by exclusion of propidium iodide. Only viable cells were collected into 5 distinct populations: GFP+ cells (Samples HC_A-D), GFP?/CD271High (Samples Mac_A-C), as well as GFP?/CD271Low/CD326+/CD146Low (Samples SC_A-B), GFP?/CD271Low/CD326+/CD146Hig (Samples GER_A-B), and GFP?/CD271Low/CD326? (Sample BM_B). Please see Sinkkonen et al 2011 PMID: 22355545 Total RNA was isolated from 5 different FACS sorted cell poulations from P3 Math1-nGFP cochlea.

ORGANISM(S): Mus musculus

SUBMITTER: Byron Hartman

PROVIDER: E-GEOD-62582 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Intrinsic regenerative potential of murine cochlear supporting cells.

Sinkkonen Saku T ST Chai Renjie R Jan Taha A TA Hartman Byron H BH Laske Roman D RD Gahlen Felix F Sinkkonen Wera W Cheng Alan G AG Oshima Kazuo K Heller Stefan S

Scientific reports 20110629

The lack of cochlear regenerative potential is the main cause for the permanence of hearing loss. Albeit quiescent in vivo, dissociated non-sensory cells from the neonatal cochlea proliferate and show ability to generate hair cell-like cells in vitro. Only a few non-sensory cell-derived colonies, however, give rise to hair cell-like cells, suggesting that sensory progenitor cells are a subpopulation of proliferating non-sensory cells. Here we purify from the neonatal mouse cochlea four different ...[more]

PMID: 22355545

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Project description:We sought to determine which gene transcripts are enriched in Wnt-responsive adrenocortical mouse cells compared to the entire adrenocortical mouse cell population in vivo. To this end, we employed transgenic reporter mice that label Wnt-responsive cells with GFP expression (TCF/Lef:H2B-GFP mice) or label all adrenocortical cells with GFP expression (Sf1:eGFP mice). GFP-positive adrenocortical cells were obtained from 6-week-old male TCF/Lef:H2B-GFP mice and Sf1:eGFP mice independently. 10 adrenals per genotype per sort were minced and digested by incubation in DMEM:F12 containing 0.1% collagenase/ 0.01% DNaseI solution for 1 h at 37Â°C. A single cell suspension was obtained following mechanical dispersion, filtration through a 40 micron nylon cell strainer, centrifugation at 1500rpm for 5 min followed by re-suspension in sterile 1X PBS containing 10% cosmic calf serum and 10Î¼g/mL Propidium iodide. 10,000-50,000 viable GFP-positive cells were isolated via FACS using a BD FACSAria III cell sorter. RNA was extracted using an RNeasy Micro Kit (Qiagen) from 4 independent sorts per genotype. cDNA were prepared according to the NuGen WT-Pico V2 kit protocol from 5 ng total RNA (Ovation PicoSL WTA System V2 P/N 3312). Biotinylated single-stranded cDNA were prepared from 3ug of cDNA (Encore Biotin Module P/N 4200-12, 4200-60, 4200-A01). Targets were assayed on the Mouse Gene ST 1.1 strip arrays using the Affymetrix Gene Atlas system (software version 1.0.4.267). One TCF/Lef:H2B-GFP array was deemed low-quality and discarded. Two-sample T-tests were used to compare the two groups of samples. We also supply a supplementary file holding the data and some statistical analysis, as well as probe-set annotation that we used at that time (users may wish to obtain new annotation though). We analyzed only 28944 probe-sets with category "main", "---", and "flmrna->unmapped" according to Affymetrix annotation. GFP-positive adrenocortical cells were obtained from 6-week-old male TCF/Lef:H2B-GFP mice and Sf1:eGFP mice independently. 10 adrenals per genotype per sort were minced and digested by incubation in DMEM:F12 containing 0.1% collagenase/ 0.01% DNaseI solution for 1 h at 37Â°C. A single cell suspension was obtained following mechanical dispersion, filtration through a 40 micron nylon cell strainer, centrifugation at 1500rpm for 5 min followed by re-suspension in sterile 1X PBS containing 10% cosmic calf serum and 10Î¼g/mL Propidium iodide. 10,000-50,000 viable GFP-positive cells were isolated via FACS using a BD FACSAria III cell sorter. RNA was extracted using an RNeasy Micro Kit (Qiagen) from 4 independent sorts per genoytpe. cDNA were prepared according to the NuGen WT-Pico V2 kit protocol from 5 ng total RNA (Ovation PicoSL WTA System V2 P/N 3312). Biotinylated single-stranded cDNA were prepared from 3ug of cDNA (Encore Biotin Module P/N 4200-12, 4200-60, 4200-A01). Targets were assayed on the Mouse Gene ST 1.1 strip arrays using the Affymetrix Gene Atlas system (software version 1.0.4.267).

Dataset Information

Intrinsic regenerative potential of murine cochlear supporting cells

Publications

Intrinsic regenerative potential of murine cochlear supporting cells.

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