Retention of progenitor cell phenotype in otospheres from guinea pig and mouse cochlea

Abstract

Background: Culturing otospheres from dissociated organ of Corti is an appropriate starting point aiming at the development of cell therapy for hair cell loss. Although guinea pigs have been widely used as an excellent experimental model for studying the biology of the inner ear, the mouse cochlea has been more suitable for yielding otospheres in vitro. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy.

Methods: Organs of Corti were surgically isolated from P3 guinea pig or mouse cochlea, dissociated and cultivated under non-adherent conditions. Cultures were maintained in serum-free DMEM:F12 medium, supplemented with epidermal growth factor (EGF) plus either basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFα). Immunofluorescence assays were conducted for phenotype characterization.

Results: The TGFα group presented a number of spheres significantly higher than the bFGF group. Although mouse cultures yielded more cells per sphere than guinea pig cultures, sox2 and nestin distributed similarly in otosphere cells from both organisms. We present evidence that otospheres retain properties of inner ear progenitor cells such as self-renewal, proliferation, and differentiation into hair cells or supporting cells.

Conclusions: Dissociated guinea pig cochlea produced otospheres in vitro, expressing sox2 and nestin similarly to mouse otospheres. Our data is supporting evidence for the presence of inner ear progenitor cells in the postnatal guinea pig. However, there is limited viability for these cells in neonatal guinea pig cochlea when compared to the differentiation potential observed for the mouse organ of Corti at the same developmental stage.

Figure 1

Images represent analyses taken at a Zeiss Axiovert 40C inverted microscope and an Axiocamera MRC5 (Zeiss, Germany) of spheres observed with phase contrast while culturing of dissociated mouse or guinea pig cochleas, with either bFGF or TGFα, as indicated. Scale bar 50 μm.

Figure 2

Indirect immunofluorescence of mouse or guinea pig otospheres from first or second passage, cultivated in the presence of either bFGF or TGFα, as indicated. The neural stem cell markers, sox2 and nestin, were used to label the cells. DAPI identifies cell nuclei. Scale bar 10 μm.

Figure 3

Images of analyses taken at a Zeiss Axiovert 40C inverted microscope and an Axiocamera MRC5 (Zeiss, Germany) of spheres observed with phase contrast while culturing of dissociated mouse or guinea pig cochleas, with either bFGF or TGFα, as indicated. P0 and P1 indicate primary culture and first subculturing, respectively. Arrows indicate otospheres obtained from guinea pig. Scale bar 50 μm.

Figure 4

Images represent analyses taken at a Zeiss Axiovert 40C inverted microscope and an Axiocamera MRC5 (Zeiss, Germany) of spheres observed with phase contrast while culturing of dissociated mouse or guinea pig cochleas, as indicated. Data shown was obtained with TGFα-supplemented medium. Similarly, otospheres cultivated in culture medium with bFGF presented the same pattern of self-renewal (not shown). All images are from passage-one cells, cultivated for one (1DIV) or four (4DIV) days in vitro. Arrows indicate otospheres. Scale bar 50 μm.

Figure 5

Indirect immunofluorescence of mouse otospheres from second passage, cultivated in the presence of bFGF, and submitted to dish adherence and cell differentiation. Myosin VIIa, a marker for hair cells, is labeled by Alexa 488 and shown in panel A. Arrows indicate plasma membrane processes, underneath which there is an enrichment of myosinVIIa. P27kip1 and Jagged 1, markers for supporting cells give the expected green staining of plasma membrane and red labeling of nuclei, respectively, shown in panels B and C. DAPI stains in blue nuclear DNA. Scale bar 10 μm.

Figure 6

Indirect immunofluorescence of mouse otospheres from second passage, cultivated in the presence of TGFα, and submitted to dish adherence and cell differentiation. Jagged 2 (panel A) and Myosin VIIa (panel B) are hair cell markers, here labeled in red and green, respectively. Arrows indicate their concentration near the plasma membrane, especially in membrane ruffles. P27kip1 labels supporting cell nuclei, as shown in panel C. DAPI identifies the cell nucleus in blue. Scale bar 10 μm.