Figure 3. Hair cell generation only from Lgr5-expressing neurospheres.

(A) Neurospheres formed by the Sox2^pos and Sox2^neg fractions incorporated BrdU. (B) Sorted Lgr5^pos and Lgr5^neg cells formed neurospheres that incorporated BrdU. (C) Neurospheres formed by the Sox2^pos and Sox2^neg fractions did not contain hair cells (Myosin VIIa). (D) Neurospheres made from sorted Lgr5^pos and Lgr5^neg cells showed no labeling for myosin VIIa. (E) Myosin VIIa-positive cells were observed upon differentiation of spheres made from the Sox2^pos cells but not the Sox2^neg cells. (F) After differentiation, neurospheres made from Lgr5^pos cells generated myosin VIIa-positive cells. No hair cells were observed upon differentiation of neurospheres made from the Lgr5^neg cells. (G) Differentiated Lgr5^pos neurospheres retained Lgr5 and Sox2 surrounding the myosin VIIa-positive cells that lost expression of Lgr5. (H) Myosin VIIa-staining cells from Lgr5^pos spheres co-stained for parvalbumin 3. (I) Cells surrounding the myosin VIIa- positive cells expressed islet1. (J) Neuropheres made from Atoh1 (negatively-sorted), or Sox2 or Lgr5 (positively-sorted) cells had similar rates of cell division. (K) Increasing capacity for hair cell differentiation was found in neurospheres made from Atoh1, Sox2 or Lgr5-sorted cells. Means ± S.E.M. are shown, n=3. Scale bars, 10 μm. (L) Secondary spheres made by sorting Lgr5^pos cells differentiated into myosin VIIa-positive hair cells. No hair cells were observed after differentiation of secondary spheres from Lgr5^neg cells. (M) Clonal spheres from single Lgr5^pos but not from single Lgr5^neg cells from cochlear sensory epithelium gave rise to hair cells upon differentiation.