Recently, the field of tissue engineering has progressed rapidly, but poor vascularization remains a major obstacle in bioengineering cell-dense tissues, limiting the viable size of constructs due to hypoxia, nutrient insufficiency, and waste accumulation. Therefore, new technologies for fabricating functional tissues with a well-organized vasculature are required. In the present study, neonatal rat cardiomyocytes were harvested as intact sheets from temperature-responsive culture dishes and stacked into cell-dense myocardial tissues. However, the thickness limit for layered cell sheets in subcutaneous tissue was approximately 80 microm (3 layers). To overcome this limitation, repeated transplantation of triple-layer grafts was performed at 1, 2, or 3 day intervals. The two overlaid grafts completely synchronized and the whole tissues survived without necrosis in the 1 or 2 day interval cases. Multistep transplantation also created approximately 1 mm thick myocardium with a well-organized microvascular network. Furthermore, functional multilayer grafts fabricated over a surgically connectable artery and vein revealed complete graft perfusion via the vessels and ectopic transplantation of the grafts was successfully performed using direct vessel anastomoses. These cultured cell sheet integration methods overcome long-standing barriers to producing thick, vascularized tissues, revealing a possible solution for the clinical repair of various damaged organs, including the impaired myocardium.

Artificially engineered tissues may have many therapeutic applications but complex tissues are hard to create in vitro. Here, Okano and colleagues report the production of functional cardiac tissue sheets with perfusable blood vessels, which increase the thickness and survival of transplanted tissue.

Background-Regenerative therapies, including myocardial tissue engineering, have been pursued as a new possibility to repair the damaged myocardium, and previously the transplantation of layered cardiomyocyte sheets has been shown to be able to improve cardiac function after myocardial infarction. We examined the effects of promoting neovascularization by controlling the densities of cocultured endothelial cells (ECs) within engineered myocardial tissues created using our cell sheet-based tissue engineering approach. Methods and Results-Neonatal rat cardiomyocytes were cocultured with GFP-positive rat-derived ECs on temperatureresponsive culture dishes. Cocultured ECs formed cell networks within the cardiomyocyte sheets, which were preserved during cell harvest from the dishes using simple temperature reduction. We also observed significantly increased in vitro production of vessel-forming cytokines by the EC-positive cardiac cell sheets. After layering of 3 cardiac cell sheets to create 3-dimensional myocardial tissues, these patch-like tissue grafts were transplanted onto infarcted rat hearts. Four weeks after transplantation, recovery of cardiac function could be significantly improved by increasing the EC densities within the engineered myocardial tissues. Additionally, when the EC-positive cardiac tissues were transplanted to myocardial infarction models, we observed significantly greater numbers of capillaries in the grafts as compared with the EC-negative cell sheets. Finally, blood vessels originating from the engineered EC-positive cardiac tissues bridged into the infarcted myocardium to connect with capillaries of the host heart. Conclusions-In vitro engineering of 3-dimensional cardiac tissues with preformed EC networks that can be easily connected to host vessels can contribute to the reconstruction of myocardial tissue grafts with a high potential for cardiac function repair. These results indicate that neovascularization can contribute to improved cardiac function after the transplantation of engineered cardiac tissues. (Circulation. 2008;118[suppl 1]:S145-S152.)

Osteoarthritis in the knees, which can be caused by meniscal defect, constitutes an increasingly common medical problem. Repair for massive meniscal defect remains a challenge owing to a lack of cell kinetics for the menisci precursors in knee joint. The synovium plays pivotal roles during the natural course of meniscal healing and contains mesenchymal stem cells (MSCs) with high chondrogenic potential. Here, we investigated whether intra-articular injected synovium-MSCs enhanced meniscal regeneration in rat massive meniscal defect. To track the injected cells, we developed transgenic rats expressing dual luciferase (Luc) and LacZ. The cells derived from synovium of the rats demonstrated colony-forming ability and multipotentiality, both characteristics of MSCs. Hierarchical clustering analysis revealed that gene expression of meniscal cells was closer to that of synovium-MSCs than to that of bone marrow-MSCs. Two to 8 weeks after five million Luc/ LacZ1 synovium-MSCs were injected into massive meniscectomized knee of wild-type rat, macroscopically, the menisci regenerated much better than it did in the control group. After 12 weeks, the regenerated menisci were LacZ positive, produced type 2 collagen, and showed meniscal features by transmission electron microscopy. In in-vivo luminescence analysis, photons increased in the meniscusresected knee over a 3-day period, then decreased without detection in all other organs. LacZ gene derived from MSCs could not be detected in other organs except in synovium by real-time PCR. Synovium-MSCs injected into the massive meniscectomized knee adhered to the lesion, differentiated into meniscal cells directly, and promoted meniscal regeneration without mobilization to distant organs.

Meiosis is a critical stage of gametogenesis in which alignment and synapsis of chromosomal pairs occur, allowing for the recombination of maternal and paternal genomes. Here we show that FK506 binding protein (Fkbp6) localizes to meiotic chromosome cores and regions of homologous chromosome synapsis. Targeted inactivation of Fkbp6 in mice results in aspermic males and the absence of normal pachytene spermatocytes. Moreover, we identified the deletion of Fkbp6 exon 8 as the causative mutation in spontaneously male sterile as/as mutant rats. Loss of Fkbp6 results in abnormal pairing and misalignments between homologous chromosomes, nonhomologous partner switches, and autosynapsis of X chromosome cores in meiotic spermatocytes. Fertility and meiosis are normal in Fkbp6 mutant females. Thus, Fkbp6 is a component of the synaptonemal complex essential for sex-specific fertility and for the fidelity of homologous chromosome pairing in meiosis.

Regenerative therapies have currently emerged as one of the most promising treatments for repair of the damaged heart. Recently, numerous researchers reported that isolated cell injection treatments can improve heart function in myocardial infarction models. However, significant cell loss due to primary hypoxia or cell wash-out and difficulty to control the location of the grafted cells remains problem. As an attempt to overcome these limitations, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cells (two-dimensional), cell sheets, to construct three-dimensional cell-dense tissues. Cell sheet transplantation has been able to recover damaged heart function. However, no detailed analysis for transplanted cell survival has been previously performed. The present study compared the survival of cardiac cell sheet transplantation to direct cell injection in a rat myocardial infarction model. Luciferase-expressing neonatal rat cardiac cells were harvested as cell sheets from temperature-responsive culture dishes. The transplantation of cell sheets was compared to the direct injection of isolated cells dissociated with trypsin-ethylenediaminetetraacetic acid. These grafts were transplanted to infarcted rat hearts and cardiac function was assessed by echocardiography at 2 and 4 weeks after transplantation. In vivo bioluminescence and histological analyses were performed to examine cell survival. Cell sheet transplantation consistently yielded greater cell survival than cell injection. Immunohistochemistry revealed that cardiac cell sheets existed over the infarcted area as an intact layer. In contrast, the injected cells were scattered with relatively few cardiomyocytes in the infarcted areas. Four weeks after transplantation, cardiac function was also significantly improved in the cell sheet transplantation group compared with the cell injection. Twenty-four hours after cell grafting, significantly greater numbers of mature capillaries were also observed in the cardiac cell sheet transplantation. Additionally, the numbers of apoptotic cells with deterioration of integrin-mediated attachment were significantly lower after cardiac cell sheet transplantation. In accordance with increased cell survival, cardiac function was significantly improved after cardiac cell sheet transplantation in comparison to cell injection. Cell sheet transplantation can repair damaged hearts through improved cell survival and should become a promising therapy in cardiovascular regenerative medicine.

IFN-λ 1, -λ 2 and -λ 3 have been discovered as the latest members of the class II cytokine family and shown to possess antiviral activity. Murine B16 melanoma and Colon26 cancer cells were transduced with mouse IFN-λ to determine whether IFN-λ possesses antitumor activity. Overexpression of IFN-λ induced cell surface MHC class I expression and Fas/CD95 Ag, induced significant caspase-3/7 activity, and increased p21Waf1/Cip1 and dephosphorylated Rb (Ser780) in B16 cells in vitro. IFN-λ expression in tumor cell lines markedly inhibited s.c. and metastatic tumor formation in vivo compared with mock transfections (p < 0.05). Moreover, IFN-λ expression induced lymphocytic infiltrates, and an Ab-mediated immune cell depletion assay showed that NK cells were critical to IFN-λ-mediated tumor growth inhibition. Hydrodynamic injection of IFN-λ cDNA successfully targeted liver metastatic foci of Colon26 cells, and moderately decreased the mortality of mice with tumors. IFN-λ overexpression in the liver increased NK/NKT cells and enhanced their tumor-killing activity, and suggested the activation of innate immune responses. Thus, IFN-λ induced both tumor apoptosis and NK cell-mediated immunological tumor destruction through innate immune responses. These findings suggested that local delivery of IFN-λ might prove a useful adjunctive strategy in the clinical treatment of human malignancies.

Neural stem/progenitor cells (NSPCs) migrate toward a damaged area of the central nervous system (CNS) for the purpose of limiting and/or repairing the damage. Although this migratory property of NSPCs could theoretically be exploited for cell-based therapeutics of CNS diseases, little is known of the mechanisms responsible for migratory responses of NSPCs. Here, we found that sphingosine 1-phosphate (Sph-1-P), a physiological lysophospholipid mediator, had a potent chemoattractant activity for NSPCs, in which, of Sph-1-P receptors, S1P 1 was abundantly expressed. Sph-1-P-induced NSPC migration was inhibited by the pretreatment with pertussis toxin, Y-27632 (a Rho kinase inhibitor), and VPC23019 (a competitive inhibitor of S1P 1 and S1P 3 ). Sph-1-P does not act as intracellular mediator or in an autocrine manner, because [ 3 H]sphingosine, incorporated into NSPCs, was mainly converted to ceramide and sphingomyeline intracellularly, and the stimulation-dependent formation and extracellular release of Sph-1-P were not observed. Further, Sph-1-P concentration in the spinal cord was significantly increased at 7 days after a contusion injury, due to accumulation of microglia and reactive astrocytes in the injured area. This locally increased Sph-1-P concentration contributed to the migration of in vivo transplanted NSPCs through its receptor S1P 1 , given that lentiviral transduction of NSPCs with a short hairpin RNA interference for S1P 1 abolished in vivo NSPC migration toward the injured area. This is the first report to identify a physiological role for a lipid mediator in NSPC migration toward a pathological area of the CNS and further indicates that the Sph-1-P/S1P 1 pathway may have therapeutic potential for CNS injuries. STEM CELLS 2007;25:115-124