Abstract: Objective To investigate the process of cell aggregation and myocardium differentiation after the P19 cells treated with the bone morphogenetic protein 2（BMP-2）. Methods P19 cells were cultivated with BMP-2 in suspension for 4 days in dishes containing a thin layer of soft agar to form cell aggregation.The aggregates were plated on gelatin-coated culture dishes, and cultured without BMP-2 for 19 days. The cultured cells were observed using a phase-contrast microscope. Immunohistochemical technique and laser scanning confocal microscope (LSCM) were used to examine the expression of α-sarcomeric actin and cardiac-specific troponin T(C-TnT) protein. The induced cells were evaluated using a transmission electron microscope. Results Cell masses appeared after P19 cells were suspension cultured with BMP-2 for 24 hours and then globular cell masses grew into a embryonic body(EBs) after 4 days. After the aggregates were plated on gelatin-coated culture dishes, the EBs were attached regular and the core was observed to be darker and had a higher cell density. After the EBs were attached for 8 hours, cells grew from the border of the EB and formed monolayered peripheral outgrowth. Central cells were undifferentiated cells, which were multilayered,smaller and had a higher nuclear-cytoplasmic ratio and grew faster. Some cells in the peripheral outgrowth became elongated, larger and radial at day 11 and expressed α-sarcomeric actin and C-TnT protein by day 19.Immunofluorescence double staining revealed that α-sarcomeric actin and C-TnT protein co-expressed in the cytoplasm. Under the transmission electron microscope, the differentiated cells derived from Pl9 cells had a single orbicularovate cell nucleus and plenty of cell organs, including chondriosomes, endoplasmic reticulum and ribosome. Paralleled myofilaments were seen in the cytoplasma or beside the edge of membrane. Conclusion The combination of B

Key words: Bone morphogenetic protein 2, Cardiomyocyte, P19cell, Immunohistochemistry