Figure 3.

Human NCPCs/SAPs Differentiate into Chromaffin-like Cells In Vitro

(A) Immunofluorescence of H9 NCPCs/SAPs differentiated with BMP4, showing co-expression of SAP marker, αTH, chromaffin marker, PNMT, and storage vesicle marker, CgB. Scale bar, 5 μm.

(B and C) Immunofluorescence count of differentiation of NCPC-4d and NCPC-6d cells with 6 days high or low BMP4 stained for αTH, NF-200kDa, and CgB (see Figures 1A and S1B). Longer initial FGF2/BMP2 exposure resulted in a higher proportion of neuronal (NF+) and a lower proportion of CgB+ SAP cells. DAPI stain was used to assess the total number of cells. N = 8 independent experiments.

(D and E) qPCR analysis of NCPC-4d and NCPC-6d cells differentiated with BMP4 and/or DP for 6 days. BMP-4 and corticosteroids increase chromaffin marker PNMT as well as SA markers. N = 4 independent experiments.

(F and G) qPCR analysis of neuronal markers RET and MYCN of NCPC-4d and NCPC-6d cells with BMP4 and/or DP for 6 days. Inclusion of corticosteroids associated with lower neuronal marker expression. N = 4 independent experiments.

Error bars represent mean ± SEM. ns, not significant, ^∗p > 0.05, ^∗∗p > 0.01, ^∗∗∗p > 0.001, ^∗∗∗∗p > 0.0001.