Elsevier

Brain Research

Volume 1091, Issue 1, 26 May 2006, Pages 282-288
Brain Research

Research Report
Prospective identification and purification of hair cell and supporting cell progenitors from the embryonic cochlea

https://doi.org/10.1016/j.brainres.2006.02.071Get rights and content

Abstract

Expression of the cyclin-dependent kinase inhibitor p27Kip1 defines a post-mitotic population of cells in the embryonic mammalian cochlea that constitutes the nascent organ of Corti. Here, we describe techniques to purify these precursors using a transgenic p27/GFP reporter and fluorescence activated cell sorting (FACS). We demonstrate that these cells express other markers of the sensory lineage, such as Sox2, and when placed in dissociated cell culture differentiate as hair cells and supporting cells. The purified sensory progenitors thus obtained provide a means of studying the process of hair cell and supporting cell differentiation in vitro, as well as providing a means of analyzing the molecular and physiological properties of this unique population of cells.

Introduction

A leading cause of sensorineural deafness in humans is the loss of mechano-sensory hair cells in the cochlea, as regeneration of lost hair cells does not occur (Chardin and Romand, 1995). In the mouse cochlea, hair cells and supporting cells become postmitotic between E12.5 and E14.5 (Ruben, 1967). Cells exiting the cell cycle form a stripe along the length of the cochlear duct termed the zone of non-proliferating cells or ZNPC (Chen and Segil, 1999); this event is dependent on the expression of the cyclin-dependent kinase inhibitor p27Kip1 (Chen and Segil, 1999, Lowenheim et al., 1999). At E14.5, p27Kip1 is expressed throughout the postmitotic prosensory domain of the cochlear epithelium prior to the onset of hair cell differentiation. In the absence of p27Kip1, cell cycle exit is delayed, and supernumerary hair cells and supporting cells are observed (Chen and Segil, 1999). p27Kip1 is rapidly down-regulated in differentiating hair cells, but supporting cells continue to express significant levels of p27Kip1 into maturity (Chen and Segil, 1999).

We sought to test the ability of newly postmitotic cells in the ZNPC to act as progenitors of new hair cells and supporting cells. By constructing transgenic animals in which fluorescent reporter genes such as GFP are expressed in specific cell populations during development, it is possible to obtain living precursor cells for analysis, including cell culture studies (Anderson et al., 1999). To this end, we modified a bacterial artificial chromosome (BAC) containing the p27Kip1 gene by inserting a GFP reporter so that it comes under the control of the p27Kip1 regulatory sequences. This construct was then used to generate transgenic mice in which the pattern of GFP expression recapitulates that of p27Kip1 in the ZNPC of the embryonic organ of Corti. We then purified GFP-expressing cells with fluorescence-activated cell sorting (FACS) from dissociated transgenic cochlear epithelial cells. Through quantitative PCR (qPCR) analysis, we found that this purified population is enriched for markers of cochlear sensory precursors, such as p27Kip1, Islet1 (Radde-Gallwitz et al., 2004), Sox2 (Kiernan et al., 2005), and Emx2 (Cecchi and Boncinelli, 2000). Finally, we found that purified GFP-expressing cells are able to generate both sensory hair cells and supporting cells in culture.

Section snippets

Generation of the p27/GFP BAC transgenic mouse

Previous results indicated that p27Kip1 is expressed in the prosensory region of the cochlear duct at E14.5 (Chen and Segil, 1999). We used bacterial homologous recombination (Yang et al., 1999) to modify a BAC clone carrying 129 kb of mouse genomic sequence in which the p27Kip1 coding region was centrally located. The BAC was first modified by inserting a cassette into the second exon of p27Kip1 after the stop codon; the cassette contained an internal ribosomal entry site and enhanced GFP (

Discussion

Sensory hair cell loss is a leading cause of deafness in humans. Sensory hair cells and supporting cells derive from a band of non-proliferating cells, the ZNPC, that runs the length of the E14.5 mouse cochlea. By histological analysis, this band is marked by the expression of p27Kip1. Here, we demonstrate, through the use of a p27/GFP BAC transgenic mouse line and flow cytometry, that the cells marked by p27Kip1 promoter activity also express other prosensory markers, and that they generate

Animals

All animal procedures were approved by the House Ear Institutional Animal Care and Use Committee (IACUC). All mice were kept in standard animal housing conditions in a 14–10 h light–dark cycle, with food and water available ad libitum. Transgenic mice were created by DNA microinjection into B6-D2 F1 (Jackson Labs) fertilized eggs; the resulting founder animals and their heterozygous transgenic male progeny were bred to non-transgenic CD1 females for at least 6 generations. Animals for timed

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    These authors contributed equally to this work.

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