Multilineage differentiation of human single-cell–derived clones. Differentiation of bone marrow (BM)–, liver (HL)– and heart (HH)–derived single-cell clones (SCCs). The top row of figures refers to a BM-derived SCC, the middle row refers to HL-derived SCC, while the bottom row refers to HH-derived SCC. The first 3 pictures of each row illustrate multiple demonstrations of the differentiation into an ectodermic derivative (neurons, top row), a mesodermic derivative (osteoblasts, middle row), and an endodermic derivative (hepatocytes, bottom row). The last 2 pictures of each row demonstrate that each clone was also able to differentiate into derivatives of the other 2 germ layers. Specifically, panel A illustrates beta3 tubulin staining in green (A488), and panel B demonstrates tyrosine hydroxylase staining in yellow (A488). Panel C demonstrates a positive immunohistochemistry staining for acetylcholine transferase. Panel D shows α-actinin (A555, red fluorescence) and ryanodine receptor (BODIPY, green fluorescence) stainings; the insert box shows at higher magnification interdigitating α-actinin and ryanodine receptor positivities. Panel E illustrates GATA4 (A488, green fluorescence in nuclei) and cytokeratin (A555, red fluorescence) labeling; the insert box documents that cytokeratin-positive cells are also GATA4 positive. Panel F reveals a positive cytochemical reaction for alkaline phosphatase activity. Panel G documents a positive von Kossa cytochemical reaction. Panel H shows the red fluorescence of tetracyclines incorporated in calcification sites. Panel I shows in purple a positive PAS reaction. Panel J documents in green GFAP (A488) and in red beta3 tubulin–positive cells (A555). Panel K reveals PAS positivity of differentiated cells. Panel L shows, in the top row, in pseudocolors (Q-LUT, scale bar) resorufin fluorescence of cell aggregates exposed to pentoxyresorufin (PR), and in the bottom row, overlay of phase contrast and red fluorescence images of the same cells; from the left to the right are depicted the following: negative control, cells exposed to PR only, and cells exposed to phenobarbital and PR. Panel M illustrates cytokeratin staining (A555, red fluorescence). Panel N shows beta3 tubulin staining in green (A488). Panel O shows von Willebrand factor in green (A488) and DiI-labeled acetylated LDL in red; insert box is a higher magnification of the marked field. DAPI was used in all fluorescence images to label nuclei in blue. (E,J,L,M) Epifluorescence and phase contrast images obtained using a live cell imaging dedicated system consisting of a Leica DMI 6000B microscope connected to a Leica DFC350FX camera (Leica Microsystems), equipped with a 63× immersion oil (numeric aperture: 1.4; E), a 40× dry (numeric aperture: 0.6; J,M), and a 10× dry (numeric aperture: 0.25; L) objective. (B,D,H,N,O) Image acquisition was carried out by a confocal laser microscope (Leica TCS-SP2), using a 63× immersion oil objective (numeric aperture: 1.40; D) and a 20× dry objectives (numeric aperture: 0.50; A,B,H,N,O). (C,F,G,I,K) Bright field images were captured using an Olympus AX70 microscope connected to an Olympus DP50 camera (Olympus, Italy). A 20× dry objective (numeric aperture: 0.70; F,G) and a 40× dry objective (numeric aperture: 0.95; C,I,K) were used for this purpose. Color temperature: 5400°K. Adobe Photoshop software was used to compose, overlay the images and to adjust contrast (Adobe).