Differentiation of embryonic stem cells into inner ear vestibular hair cells using vestibular cell derived-conditioned medium

Abstract

Vestibular hair cells (V-HCs) in the inner ear have important roles and various functions. When V-HCs are damaged, crippling symptoms, such as vertigo, visual field oscillation, and imbalance, are often seen. Recently, several studies have reported differentiation of embryonic stem (ES) cells, as pluripotent stem cells, to HCs, though a method for producing V-HCs has yet to be established. In the present study, we used vestibular cell conditioned medium (V-CM) and effectively induced ES cells to differentiate into V-HCs. Expressions of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5) were significantly increased in ES cells cultured in V-CM for 2 weeks, while those were not observed in ES cells cultured without V-CM. On the other hand, the cochlear HC-related marker Lmod3 was either not detected or detected only faintly in those cells when cultured in V-CM. Our results demonstrate that V-CM has an ability to specifically induce differentiation of ES cells into V-HCs.

Keywords: Conditioned medium; Differentiation; Embryonic stem cells; Hair cells; Inner ear; Vestibular.

Fig. 1

Preparation of vestibular cells (VCs) and conditioned medium, and in vitro differentiation procedure. (A) Murine utricles were isolated from the inner ears of postnatal day 4 (PD4) C57BL/6 mice using a microdissection method. Isolated utricles were confirmed using RITC-labeled phalloidin. (B) Vestibular cells (VCs) showing outgrowth from utricles were obtained and cultured in ES-DM. Asterisks indicate utricle attachment to the dish. Scale bar = 50 μm. (C) Conditioned medium (CM) obtained from VCs cultured in ES-DM for 24 h was collected, then centrifuged and filtrated, and used as VC conditioned medium (V-CM). (D) In vitro hair cell differentiation procedure.

Fig. 2

Cell morphology and GFP expression of Math1-GFP ES cell-derived EBs cultured in ES-DM or V-CM. (A) Cell morphology and GFP expression of Math1-GFP ES cell-derived EBs cultured in ES-DM or V-CM for 2 weeks. Scale bar = 100 μm. (B) Flow cytometrical analysis of Math1-GFP ES cell-derived EBs cultured in ES-DM or V-CM for 2 weeks. (C) GFP positivity of Math1-GFP ES cell-derived EBs cultured in ES-DM or V-CM for 2 weeks.

Fig. 3

Gene expression analysis of hair cell-related markers in Math1-GFP ES cell-derived EBs cultured in ES-DM or V-CM. Gene expressions of Math1, Myosin6, and Brn3c, (HC-related markers), Lmod3 (cochlear HC-related marker), and Dnah5 (vestibular HC-related marker) by Math1-GFP ES cell-derived EBs cultured in ES-DM or V-CM for 2 weeks were examined using real-time RT-PCR. Values were normalized to that of β-actin expression, used as an endogenous control. *p < 0.05.

Fig. 4

Immunocytochemical analysis of hair cell-related markers expressed by Math1-GFP ES cell-derived EBs cultured in ES-DM or V-CM. The expressions of HC-related markers in Math1-GFP ES cell-derived EBs cultured in ES-DM or V-CM for 2 weeks were examined with an immunocytochemical method. Neither HC-related markers nor Math1-derived GFP were detected in EB outgrowths cultured in ES-DM, whereas most of the Math1-derived GFP positive cells in EB outgrowths cultured in V-CM simultaneously expressed Myosin6, Brn3c, and Dnah5. On the other hand, no Lmod3-immunopositive cells were detected in EB outgrowths cultured in V-CM, while Math1-derived GFP positive cells were detected. Scale bar = 100 μm.