To investigate the role of the HOX-like homeoprotein PDX1 in the formation and maintenance of the pancreas, we have genetically engineered mice so that the only source of PDX1 is a transgene that can be controlled by the application of tetracycline or its analogue doxycycline. In these mice the coding region for the tetracyclineregulated transactivator (tTA off) has replaced the coding region of the endogenous Pdx1 gene to ensure correct temporal and spatial expression of the regulatable transactivator. In the absence of doxycycline, tTA off activates the transcription of a bicistronic transgene encoding PDX1 and an enhanced green fluorescent protein reporter, which acts as a visual marker of transgene expression in living cells. Expression of the transgene-encoded PDX1 rescues the Pdx1-null phenotype; the pancreata of these mice develop and function normally. The rescue is conditional; doxycycline-mediated repression of the transgenic Pdx1 throughout gestation recapitulates the Pdx1 null phenotype. Moreover, application of doxycycline at mid-pancreogenesis blocks further development. Adult animals of the rescue genotype that were treated with doxycycline for 3 weeks shut off Pdx1 expression, decreased insulin production, and lost the ability to maintain glucose homeostasis. These results demonstrate the feasibility of controlling the formation of an organ during embryogenesis in utero and the maintenance of the mature organ through the experimental manipulation of a key developmental regulator.

Novel technologies for creating three-dimensional constructs with complex shapes would be highly useful in tissue engineering. In the present study, tubular structures were constructed using magnetic force. Magnetite nanoparticles in cationic liposomes were taken up by target cells. The magnetically labeled cells were seeded onto ultralow-attachment plates, and a magnet was placed under the wells. After 24 h of culture, the magnetically labeled cells formed a cell sheet. Subsequently, when a cylindrical magnet was rolled onto the cell sheet, the cell sheet was attracted to the magnet and formed a tube around it. The magnet was then removed, leaving behind a tubular structure. Two types of tissue were used to create tubular structures: urinary tissue, consisting of a monotypic urothelial cell layer; and vascular tissue, consisting of heterotypic layers of endothelial cells, smooth muscle cells, and fibroblasts. The present results suggest that this novel methodology using magnetite nanoparticles and magnetic force, which we have termed "magnetic force-based tissue engineering" (Mag-TE), is a promising approach to constructing tissue-engineered tubular structures.

Novel technologies to establish three-dimensional constructs are desired for tissue engineering. In the present study, magnetic force was used to construct multilayered keratinocyte sheets and harvest the sheets without enzymatic treatment. Our original magnetite cationic liposomes, which have a positive surface charge in order to improve adsorption, were taken up by human keratinocytes at a concentration of 33 pg of magnetite per cell. The magnetically labeled keratinocytes (2x10(6) cells, which corresponds to 5 times the confluent concentration against the culture area of 24-well plates, in order to produce 5-layered keratinocyte sheets) were seeded into a 24-well ultralow-attachment plate, the surface of which was composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged. A magnet (4000 G) was placed under the well, and the keratinocytes formed a five-layered construct in low-calcium medium (calcium concentration, 0.15 mM) after 24 h of culture. Subsequently, when the five-layered keratinocytes were cultured in high-calcium medium (calcium concentration, 1.0 mM), keratinocytes further stratified, resulting in the formation of 10-layered epidermal sheets. When the magnet was removed, the sheets were detached from the bottom of the plates, and the sheets could be harvested with a magnet. These results suggest that this novel methodology using magnetite nanoparticles and magnetic force, which we have termed "magnetic force-based tissue engineering" (Mag-TE), is a promising approach for tissue engineering.

Novel technologies to establish three-dimensional, in vivo-like tissue consisting of various types of cells are required for tissue engineering. We applied magnetic force to construct a heterotypic, layered coculture system of rat hepatocytes and human aortic endothelial cells (HAECs) that was not limited by cell type. Magnetite cationic liposomes carrying a positive surface charge to improve adsorption accumulated in HAECs at a concentration of 38 pg of magnetite per cell. Magnetically labeled HAECs specifically accumulated onto hepatocyte monolayers at sites where a magnet (4000 G) was positioned, and then adhered to form a heterotypic, layered construct with tight and close contact. This cocultured construct significantly (p < 0.05) enhanced albumin secretion by hepatocytes compared with that in homotypic cultures of hepatocytes or heterotypic cocultures of hepatocytes and HAECs without magnets. These results suggest that this novel use of magnetite nanoparticles and magnetic force, which we refer to as "magnetic force-based tissue engineering" (Mag-TE), offers a major advancement in tissue engineering.