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• Figure S1 - Percentage of cells positive for hair cell and neuronal cell markers at step 2 is higher than that at step 1. CSCs were incubated with SERB for 14 days (step 1) or with SERB for 14 days and followed by with no SERB for 7 days (step 2). Cells at days 14 and 21 were harvested for hair cell markers (Math1, myosin VIIa, and Espin) and neuronal cell markers (â3-tubulin, MAP2, and GFAP) by FACS. It was found that positive cells for hair cell and neuronal cell markers were either higher in numbers or stronger in intensity in step 2 than in step 1 (A). GFAP positive 7 days after withdrawal of SERB are suggestive of the possible existence of astrocytes because GFAP is thought as a marker for astrocytes in differentiated cells. Immunohistochemistrically, CSCs differentiated into myosin VIIa positive (b, red) and â3-tubulin positive (c, green) cohorts 7 days after withdrawal of SERB in a representative culture (B). bar=50 ìm in (a) applying to (a-d).
• Figure S2 - Hair cell and neuronal differentiation is more profound in step 2 than in step 1 morphologically. Hair cell morphology was obvious induced in step 2 (a-c) in comparison with those in step 1 (e-g). Similarly, neuron cell morphology in step 2 (i-k) was more mature than that in step 1 (m-o). Newly differentiated hair cells are hallmarked by the expression of myosin VIIa (a & e) and Math1 (b & f) whereas nascent neurons are identified by the expression of â3-tubulin (I & m) and MAP2 (j & n). bar=10 ìm. Non-specific IgG control showed negative staining for immunohistochemistry (right column, d, h, i, & p).
• Figure S3 - Percentage of cells positive for hair cell markers at step 2 is higher in SERB-treated than in EGF+bFGF- and basal media-treated. CSCs were incubated with SERB for 14 days followed by with no SERB for 7 days (step 2). Cells at days 21 were harvested for quantitative analysis of hair cell markers (myosin VIIa and Math1) by FACS as described in the Methods section. It was found that numbers of the positive cells for hair cell markers were significantly higher in SERB-treated cells than in EGF+bFGF- or basal media (Ctrl)-treated cells (A, *p<0.05). Similarly, the mRNA transcripts for myosin VIIa (myo7a) and Math1 in SERB-treated cells were relatively higher than those in EGF+bFGF-treated cells by semi-quantitative RT-PCR analysis (B). It is noted that RA regulated the expression of the Math1 mRNA transcripts more than other individual factors or SERB. Ctrl in (B), RT-PCR control (omitting reverse transcriptase); GAPDH, RNA sample loading control.