Culture of cells recruited into the matrigel sponges by VEGF.

<p>After 1 week <i>in vivo</i> the MG was removed and the cells infiltrating the matrigel sponge were plated out at a low density and allowed to form colonies over 24 hrs. (A) Phase contrast image of Matrigel extracted after 1 week <i>in vivo</i>. It is infiltrated by numerous rounded cells that stained with Giemsa solution (A1). (B) Phase contrast image of small colonies after 24 hrs from seeding formed by rounded cells which stained with Giemsa (C) incorporate BrdU in their nuclei (arrowheads in D). (E–G) Double labelling experiments showing the expression in these cells of the myogenic marker MyoD (green in E–G) and the endothelial markers FLK-1 (red in E), VE-cadherin (red in F), and CD34 (red in G). (H) TEM micrograph of precursors cells showing an undifferentiated phenotype with large nucleus (n) and scarce cytoplasm. (I) Phase contrast image of a single colony that was picked with a micropipette and clonally expanded for a total of 3 days in vitro. (J–L) Double labelling experiments of clonal colonies showing the expression of MyoD (green in J–L) and the endothelial markers FLK-1 (red in J), VE-cadherin (red in K) or CD34 (red in L). (M) Clonal expansion of single colonies in phase contrast after 8 days in vitro. Rounded (arrowhead) and spindle-shape cells (arrow) that stained with Giemsa solution (N) are visible. (O) Positivity for BrdU incorporation is detectable only in the nuclei (arrowhead) of the rounded cells. Double immunofluorescence experiments detect the expression of CD34 (red in P) and MyoD (green in P) in the cultured cells. These cells are desmin⁺ as well (red in Q). (R) TEM image showing thick filaments (arrowheads) in differentiating elongated cells. Phase contrast image (S) and Giemsa staining (T) of spindle-shape cells that dominate the clonal cultures after 20 days in vitro. The differentiating spindle shaped cells are BrdU⁻ (U), CD34⁺ (green in V) and skeletal MyHC⁺ (red in Z). (W) TEM photomicrograph showing a well differentiated muscle fibre with contractile material clearly organized into sarcomeres (arrowhead in W). Nuclei are counterstained with DAPI (blue in Q, V, Z). Bars in A–D: 100 µm; Bars in E–G: 20 µm Bars in I–L, V, Z 15 µm; Bars in M–Q, S–U: 10 µm; Bars in H, R, W: 400 nm.