Figure 5.

Chondrocyte, Endothelial Progenitor Cell, and Definitive Endoderm Differentiation Following BA Pretreatment

(A) Schematic depicting the micromass culture protocol utilizing E8 (blue), E6 (green), or BA (orange) pretreatment.

(B) Sulfated glycosaminoglycan (left) and hydroxy-proline (right) levels were elevated in BA-treated cells compared with both E6 and E8 in two iPSC lines and H9 hESCs after 7 days of micromass culture.

(C) Alcian blue staining for proteoglycan production after 7 days of micromass culture. Scale bar, 2 mm.

(D) Flow cytometry analysis of cell surface marker expression after integration of E8-, E6-, or BA-pretreated cells into an EPC differentiation protocol. Isotype immunoglobulin antibody was used as a negative control.

(E) Schematic representation of definitive endoderm induction protocol.

(F and G) Quantification (F) of SOX17⁺ and FOXA2⁺ cell populations based on immunofluorescent staining in (G). Scale bar, 100 μm.

(H) Gene expression analysis of DE genes SOX17, FOXA2, GATA6, visceral endoderm gene AFP, and mesendoderm/mesoderm gene MIXL1, following the 96-h DE induction protocol.

Errors bars represent SEM, n = 3 independent experiments, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 for (B), (F), and (H). See also Figure S3.