Light and electron microscopy of stage-specific features of the transdifferentiation of mouse Meckel's cartilage chondrocytes in vitro.

The changes in the morphological characteristics of Meckel's cartilage cells in culture can be divided into five stages during their transdifferentiation to cells with an osteocyte-like phenotype, as demonstrated in this study by light and electron microscopy. Chondrocytes were isolated from day-17 embryonic mice by enzymatic digestion and inoculated at a density of 1 x 10(4) cells. The cultures were incubated at 37 degrees C and cells were harvested every week for 4 weeks and processed for light and electron microscopy, as well as an immunohistochemical analysis of type I and II collagens. The primary cultures were characterized by fibroblastic cells (stage 1) around days 2-4. Polygonal cells (stage 2) expanded on a poorly formed extracellular matrix on day 7, and then they differentiated into large, round cells (stage 3) which began to form cellular nodules. The large, round cells contained many vacuoles, which were rapidly released into the pericellular space with a significant resultant decrease in cell size. Immunostaining showed that the extracellular matrix was occupied predominantly by type II collagen after 2 weeks in culture. However, the type II collagen was gradually replaced by type 1 collagen. Cells that formed multiple layers gradually changed into small, round cells (stage 4) at the tops of nodules and ultimately expressed an osteocytic phenotype (stage 5) after further transdifferentiation.