Figure 3. Effect of known DYRK1A and GSK3ß inhibitors and ID-8 analogues on induction of PAX6 positive cells by dual SMAD inhibition.
(A) structures of known DYRK1A inhibitors studied. (B) percentage of PAX6 positive cells present at Day 16 in cultures of pluripotent cells, cultures of neural progenitors induced by dual SMAD inhibition (DMSO), and cultures subjected to neural induction in the presence of DYRK1A inhibitors at a dose of 0.5 µM. Error bars, SD; **p<0.05, *p<0.0.01. (C) percentage of PAX6 positive cells present at Day 16 in cultures of pluripotent cells, cultures of neural progenitors induced by dual SMAD inhibition (DMSO), or cultures subjected to neural induction in the presence of ID-8 analogues. All compounds were tested at 0.5 µM. Error bars, SD; **p<0.05, *p<0.0.01. (D) indirect immunofluorescence micrographs showing stem cell cultures subjected to dual SMAD inhibition alone or in combination with 5.0 µM ID-8, 5.0 µM compound 28, or 3.0 µM CHIR 99021 and stained with antibodies to POU5F1, PAX6 or NESTIN (all red) and DAPI nuclear counterstain (dark blue). (E) flow cytometry analyses of stem cell surface marker (GCTM-2 antigen and TG30 anti-CD9 antibody) expression in cultures subjected to dual SMAD inhibition alone or in combination with 5.0 µM ID-8, 5.0 µM compound 28, or 3.0 µM CHIR 99021. B and C, studies carried out with HES3 (PAX6mCherry) cell line; D-E, WA09.