Skip to main content
. 2024 Sep 8;26:749–759. doi: 10.1016/j.reth.2024.08.024

Fig. 1.

Fig. 1

Optimization of iNCCs induction and purification methods. A. The diagram illustrates the iNCC induction and purification method. SB, SB431542. CHIR, CHIR99021. B. The histogram represents the results from flow cytometry measurements of the heterogeneous cell population following NCC induction, stained with isotype control (a) or CD271 antibody (b). Among the CD271 positive cells, the populations with a fluorescence intensity of 104 (marked with a line) or higher were defined as high CD271 expression (CD271high) cells. C. Morphology of colonies on day 14 when iNCCs were induced from iPSCs under various CHIR concentrations (0.3 μM (a), 0.6 μM (b), 0.9 μM (c)), along with the percentage of CD271high cells induced at each concentration (d). Statistical significance was determined using an unpaired t-test. ∗p<0.05. Data are represented as the mean ± SD, n = 3. Scale Bar: 500 μm. D. Representative microscopic images of iPSCs (201B7) cultured in StemFit AK03 N supplemented with either LN211 or LN511-E8 at their respective concentrations. Arrowheads indicate the colonies. Scale Bar: 500 μm. E. The percentage of CD271high cells in the first passage after culture with each scaffold. The low dose of LN511-E8, LN211, and VN was 0.6 μL/mL, and the high dose was 6 μL/mL. The low dose of FN was 15 μL/mL, and the high dose was 30 μL/mL. F. The relative gene expression of NCC Marker Genes (SOX10, TFAP2A, SOX9, TWIST1) and pluripotency marker (OCT3/4) in iNCCs purified using LN211 compared with the expression of iPSCs (201B7). The expression levels of each gene were normalized by that of the housekeeping gene, β-actin (ACTB). Data are represented as the mean ± SD, n = 3. G. The histograms display the results from flow cytometry measurements of various iPSC-derived cells pre (upper) and post (lower) iNCCs purification (left: 201B7, middle: 1210B2, right: 1231A3). Cell populations with a fluorescence intensity of 104 or above (the right side of the vertical line) are classified as CD271high cells. The data were obtained with N = 1.