Fig 2: Generation of neural progenitors for transplantation and the neural markers of MGE neural progenitors during the process of differentiation. (a) Representative images were immunoreactive for neuroepithelium marker PAX6 and NSC marker Nestin on day 4. (b) Coimmunostaining SOX1 and PAX6 at day 10. (c) Differentiated hESCs expressed forebrain marker, FOXG1, but no spinal cord marker, HOXB4. (d) Coexpressing MGE neural progenitor marker NKX2.1 and FOXG1 on day 14. By 49 days in culture, neurons were positive for (e) GABA and (f) CHAT. Scale bars = 50 μm.

Fig 3: Effect of Pro-Hyp on the mRNA expression levels of Foxg1, Runx2, osterix, Col1α1, osteocalcin and Foxo1 in Foxg1-knockdown cells. Foxg1 was knocked down in MC3T3-E1 cells, which were then cultured with or without 0.1 mM Pro-Hyp for 2 days. After incubation, total RNA was extracted and reverse transcribed, and real-time PCR was carried out. The mRNA expression levels of Foxg1 (a), Runx2 (b), osterix (c), Col1α1 (d), osteocalcin (e) and Foxo1 (f) are shown. Data are presented as means ± SD (n = 4). Bars not sharing a letter differ, p < 0.05

Fig 4: Characterization of otic neural progenitors (ONPs) derived from hESCs before and after purification. (a) Immunofluorescence images demonstrating the expression of PAX6 and SIX1 in eONPs before and after purification. In the ONP stage, PAX6+ cells occupy almost 80% of cells before purification, and PAX6+ cells reached 92% ± 2% after purification. About 20% of the cells expressed SIX1 before purification, and SIX1+ cells reached 80% ± 3% after purification. Scale bars: 100 μm. (b) and (c) Quantification of PAX6 (b) and SIX1 (c) expression levels in eONPs before and after purification using collagenase IV, as determined by immunocytochemistry staining (n = 4; ****p < 0.0001; **p < 0.01; t-test). (d) Immunofluorescence staining of eONPs for various neural progenitor markers: FOXG1 (a), PAX2 (b), Brn3A (c), NESTIN (d), PAX8 (e), and TUJ1 (f). Scale bars: 100 μm. (e) Diagram of quantification illustrating the expression proportions of ONP markers (FOXG1, PAX2, Brn3A, TUJ1, NESTIN, and PAX8) as determined by immunostaining of purified eONPs at day 19 of differentiation. (n = 4).

Fig 5: Chromatin immunoprecipitation profiling of Foxg1 protein enrichment at L1 elements. (A,B) Schematics of the protocols and lentiviral vectors employed are on the left. Graphs show results from early- (A) and mid- (B) neuronogenic cultures, set according to type I (A) and II (B) protocols. Cultures constitutively overexpressing Foxg1 (Foxg1-OE) or a Plap-OE control) are driven by the pPgk1 promoter. Chromatin immunoprecipitation was performed using anti-Foxg1 antibody and control IgG. PCR quantitation of pan-L1 diagnostic amplicons L1.orf2 and L1.3′UTR, and family-specific L1.5′UTR amplicons .A, .Gf and .Tf is shown. Results are normalized against input chromatin. Error bars indicate s.e.m. Statistical significance of results was evaluated using a one-tailed unpaired t-test. n=number of biological replicates, i.e. independently cultured and engineered cell aliquots originating from a common neural precursor pool.