CA-PKA enhances EC differentiation from Flk1⁺ vascular progenitors. (A) Experimental system for PKA activation. An embryonic stem (ES) cell line expressing constitutive active (CA) form of PKA by tetracycline-inducible expression system (Tet-Off) was established. Doxycycline (Dox) was added during the first 4.5-day culture of ES cell differentiation to Flk1⁺ cells. Flk1⁺ cells were sorted by magnetic cell sorting (MACS) and subjected to two-dimensional culture on collagen-coated dishes or three-dimensional culture in collagen gel, and were cultured in the presence or absence of Dox (1 μg/mL). (B-D) Two-dimensional culture with DM, at Flk-d3. (B) Double immunostaining for CD31 (purple) and αSMA (brown). (Left panel) Dox (1 μg/mL) treatment. (Right panel) Dox-free. Culture with DM alone. Scale bar represents 100 μm. (C) Flow cytometry for EC markers, CD31 and VE-cadherin. Percentages of CD31⁺/VE-cadherin⁺ ECs in total Flk1⁺ cell–derived cells are indicated. (D) Fluorescent staining for CD31 (green) and DAPI (blue). (Left panels) Dox (1 μg/mL) treatment. (Right panels) Dox-free. Flk1⁺ cells stimulated with vehicle (top panels) or VEGF (50 ng/mL; bottom panels). Scale bars represent 250 μm. (E-I) Three-dimensional culture of Flk1⁺ cell aggregates in type I collagen gel with DM alone. (E) Phase-contrast images after 5-day culture. (Left panel) Dox (1 μg/mL) treatment. (Right panel) Dox-free. Scale bars represent 100 μm. (F) In-gel double immunostaining for CD31 (purple) and αSMA (brown) in Dox-free condition. (Left panel) Gross appearance of vascular structure. (Right panel) Higher magnification view. αSMA⁺ cells attached to CD31⁺ EC tube structure are observed (arrows). Scale bars represent 100 μm. (G-H) Cross-section of three-dimensional culture in Dox-free condition. (G) Hematoxylin-eosin staining. (H) Double immunostaining for CD31 (brown) and αSMA (red). Right panels correspond to boxed regions. Scale bars represent 250 μm. αSMA⁺ cell attached to CD31⁺ EC tube structure is observed (arrow). (I) Confocal microscopic analysis of vascular structure. Double fluorescent staining for CD31 and αSMA in Dox-free condition. (Left panel) CD31 (green). (Middle panel) αSMA (red). (Right panel) Merged image. αSMA⁺ cell attached to CD31⁺ EC tube structure is observed (arrow). CD31⁺ cells formed true lumen (green) with attached mural cells (red) shown in xz image. Dashed line indicates sliced position. Scale bars represent 100 μm.