Figure 8. Commitment ability of stem cells present in the HDL.

(A) Osteogenic commitment. Immunofluorescence confirms positive staining for osteonectin (red) in both 2D cultures (scale bar = 20 µm) and 3D cultures (scale bar = 50 µm). Real-time PCR detects the expression of osteopontin (OPN), osteonectin (ON), osteocalcin (OCN), and type I collagen (COL1A1), which are typical markers of osteogenic commitment. (B) Adipogenic commitment. ORO staining confirms lipid deposition inside the cytoplasm (red) in 2D cultures (scale bar = 50 µm). SEM analysis of 3D cultures reveals the typical adipogenic round shape of cells (scale bar = 10 µm). Genes related to the adipogenic phenotype, such as PPARG, LPL, GLUT4, and ADIPOQ, are expressed. (C) Chondrogenic commitment. In the 3D cultures, the cells are organized into their typical clusters, as shown by SEM analysis (scale bar = 50 µm), and only express type II collagen (COL2A1), as detected by real-time PCR. (D) Neuronal commitment. Immunofluorescence confirms positive staining for TUBB3 (red) in both 2D cultures (scale bar = 40 µm) and 3D cultures (scale bar = 100 µm). High expression of TUBB3 and low expression of GFAP are evident. (E) Glial commitment. Immunofluorescence confirms positive staining for GFAP in both 2D cultures (red, scale bar = 20 µm) and 3D cultures (green, scale bar = 100 µm), where significant GFAP mRNA expression is also detectable. (F) Endothelial commitment. Immunofluorescence confirms positive staining for VWF (red) in 2D cultures (scale bar = 20 µm) and for CD31 (green) in 3D cultures (scale bar = 100 µm). In the 3D scaffolds, the cells are also able to organize into micro-capillary vessels. Gene expression of endothelial markers, such as CD31 and VEGF, is clearly detectable.