While the syncytial nature of myofibers may render skeletal TH-302 CYP17 inhibitor muscle innately amenable to a cell fusion based therapy, a small number of other therapeutically interesting cell types including cardiomyocytes and hepatocytes also naturally exist in multinucleated states . Furthermore, these cell types as well as INCB28060 customer reviews others including Purkinje neurons and renal proximal tubule epithelial cells are known to tolerate fusion and exist as heterokaryons following bone marrow transplantation . However, as in the case of skeletal muscle, these fusion events are extremely infrequent and therapeutic effects are only observed in rare cases where heterokaryons exhibit a growth advantage over resident cells . Therefore, in order to treat the vast majority of pathologies in which positive selection does not occur, the efficiency of the fusion process must be increased. Clearly cell surface markers of suitable specificity must be validated for each cell type. However, if such markers can be identified, targeted cell fusion may represent a novel therapeutic approach to a number of degenerative diseases. MEF Ha7/F and MEF Ha7 cells were maintained in DMEM supplemented with 10% fetal bovine serum and 30 mM bmercaptoethanol. Prior to transplantation, cells were trypsinized, resuspended in PBS and counted. 16105 cells from each population were then intramuscularly injected into 7-week-old male C57BL/6 recipients utilizing a 26-gauge needle. In the damage model, cells were co-injected with 10 mL of notexin . Muscle tissue was then allowed to heal for one week prior to analysis. As a positive control for the detection of GFP-positive cells in other organs, 16107 whole bone marrow cells from a GFP-positive mouse were injected intravenously into wild type mice and these recipients were harvested three hours later. For analysis, mice were first terminally anesthetized with avertin, then perfused with PBS containing 10 mM EDTA and finally perfused with 4% PFA in PBS. All lower leg muscles as well as the spleen, lung and liver were then removed from recipients and post-fixed in 4% PFA overnight prior to overnight cryoprotection in 20% sucrose. All tissues were then embedded and cut into 20 mm 10 mm or 5 mm sections . Muscle sections were stained with rabbit anti-mouse laminin overnight at 4uC, followed by a one hour incubation with goat anti-rabbit Alexa 568 at room temperature. Stained muscle tissues were analyzed by confocal microscopy using a Nikon C1 laser scanning confocal microscope and images are presented as maximum intensity projections of Z stacks of individual optical sections.