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Fig. 6. Microinjection of hMSCs along the Wolffian duct (WD). (a) E11.5 rat embryos were microinjected with DiI-labeled hMSCs and observed by using a fluorescent microscope . Injected hMSCs are indicated by the arrow. To examine whether the microinjection was correctly performed at the budding site, adjacent sections of the Wolffian duct were subjected to hematoxylin/eosin staining (b) and in situ hybridization by using human specific AluI/II sequences as probe (c). AluI/II-positive human cells are located around the Wolffian duct.

Fig. 7. Y chromosome FISH of single-cells lysate generated from neometanephros. After relay culture, neogenerated metanephroi containing male hMSCs were digested in collagenase, and single cells were subjected to FISH by using probes for the human and rat Y chromosome as described (1). The hybridization solution containing a human- and rat-painting Y probe, and hybridization buffer were purchased from Cambio (Cambridge, U. K.). The majority of cells contained only one Y chromosome signal (yellow arrows) and a negligible number of cells (< 0.1%) were doubly positive, suggesting that the occurrence of cell fusion during relay culture is rare. Six cells appear in this field and each cell has one Y chromosome.

Fig. 8. Distribution of donor hMSCs during organ culture. During metanephric organ culture, microinjected transplanted cells were traced by using a fluorescent microscope with a rhodamine filter set. DiI-positive cells divided and migrated into the metanephric rudiment are shown. White dashes indicate the outlines of metanephric rudiment.

1. Yokoo, T., Ohashi, T., Utsunomiya, Y., Shen, J.-S., Hisada, Y., Eto, Y., Kawamura, T. & Hosoya, T. (2001) Blood 98, 57-64.

Movie. 1. The whole-embryo culture system is shown in this movie. Rat embryos, together with the yolk sac, amnion, and chorioallantoic placenta, are cultured in the rotating bottle, which is continuously supplied with a programmed concentration of oxygen. Viability of the embryos can be confirmed by heart beat, whole-body blood circulation, and general morphology.