Although mesenchymal stem cells (MSCs) are the natural source for bone regeneration, the exact mechanisms governing MSC crosstalk with collagen I have not yet been uncovered. Cell adhesion to collagen I is mostly mediated by three integrin receptors – α1β1, α2β1 and α11β1. Using human MSC (hMSC), we show that α11 subunit exhibited the highest basal expression levels but on osteogenic stimulation, both α2 and α11 integrins were significantly upregulated. To elucidate the possible roles of collagen-binding integrins, we applied short hairpin RNA (shRNA)-mediated knockdown in hMSC and found that α2 or α11 deficiency, but not α1, results in a tremendous reduction of hMSC numbers owing to mitochondrial leakage accompanied by Bcl-2-associated X protein upregulation. In order to clarify the signaling conveyed by the collagen-binding integrins in hMSC, we analyzed the activation of focal adhesion kinase, extracellular signal-regulated protein kinase and serine/threonine protein kinase B (PKB/Akt) kinases and detected significantly reduced Akt phosphorylation only in α2- and α11-shRNA hMSC. Finally, experiments with hMSC from osteoporotic patients revealed a significant downregulation of α2 integrin concomitant with an augmented mitochondrial permeability. In conclusion, our study describes for the first time that disturbance of α2β1- or α11β1-mediated interactions to collagen I results in the cell death of MSCs and urges for further investigations examining the impact of MSCs in bone conditions with abnormal collagen I.

Objective-Cord blood-derived human endothelial colony-forming cells (ECFCs) bear a high proliferative capacity and potently enhance tissue neovascularization in vivo. Here, we investigated whether the leading mechanism for the functional improvement relates to their physical vascular incorporation or perivascular paracrine effects and whether the effects can be further enhanced by dual-cell-based therapy, including mesenchymal stem cells (MSCs). Methods and Results-ECFCs or MSCs were lentivirally transduced with thymidine kinase suicide gene driven by the endothelial-specific vascular endothelial growth factor 2 (kinase insert domain receptor) promoter and evaluated in a hindlimb ischemia model. ECFCs and MSCs enhanced neovascularization after ischemic events to a similar extent. Dual therapy using ECFCs and MSCs further enhanced neovascularization. Mechanistically, 3 weeks after induction of ischemia followed by cell therapy, ganciclovir-mediated elimination of kinase insert domain receptor ϩ cells completely reversed the therapeutic effect of ECFCs but not that of MSCs. Histological analysis revealed that ganciclovir effectively eliminated ECFCs incorporated into the vasculature. Conclusion-Endothelial-specific suicide gene technology demonstrates distinct mechanisms for ECFCs and MSCs, with complete abolishment of ECFC-mediated effects, whereas MSC-mediated effects remained unaffected. These data strengthen the notion that a dual-cell-based therapy represents a promising approach for vascular regeneration of ischemic tissue. (Arterioscler Thromb Vasc Biol. 2012;32:e13-e21.)Key Words: angiogenesis Ⅲ coronary heart disease Ⅲ endothelium Ⅲ ischemia Ⅲ peripheral arterial disease D iseases of the cardiovascular system remain the leading causes of mortality and still account for more deaths than cancer, chronic lower respiratory diseases, and accidents together. 1 Regenerative medicine using stem and progenitor cells from different sources is a rapidly growing area of research aiming for repair or replacement of injured tissues. Some previous cell-based therapies using adult-derived stem and progenitor cells showed significant, albeit modest functional improvements and, therefore, did not fulfill high expectations. 2 On the other hand, ethical controversy and immunologic barriers over pluripotent embryonic stem cells hindered their therapeutic application, whereas induced pluripotent cells are still in their infancy and require comprehensive characterization and complete depletion of teratogenic cells before their clinical use. Still, the future use of induced pluripotent cell-derived, patientspecific, highly proliferative ECFCs raises the question of potential security measures, such as the use of suicide genes to stop excessive proliferation.To date, cell therapy based on the use of autologous endothelial progenitor cells (EPCs) still remains a safe, promising, and innovative therapeutic approach. These cells are capable of enhancing neovascularization after ischemic insults, including limb ischemia, acute myocar...

The purpose of this study was to establish inducible transgene expression in pigs, a model organism with great promise for experimental physiology and translational medicine, using the binary tet-on system. This expression system is activated by doxycycline (dox) via the tet-controlled transactivator (TA). Binding of TA to the transactivator response element (TRE) results in transcription of downstream genes. First, we cloned transgenic founder pigs expressing TA under the control of the CMV enhancer/chicken β-actin promoter (CAG). Then, cells from CAG-TA transgenic founders were nucleofected with TRE-controlled expression vectors for either porcine cytotoxic T-lymphocyte associated antigen 4-Fc domain of immunoglobulin G1 (CTLA-4Ig) or soluble receptor activator of NF-κB ligand (RANKL), and double-transgenic offspring were cloned. Dox administration resulted in a dose-dependent increase in expression of CTLA-4Ig or RANKL, in nucleofected cells and in transgenic pigs, while in the absence of dox, the levels of both proteins were below the detection limit. Inducible transgene expression was reproduced in double-transgenic offspring generated by cloning or breeding. Our strategy revealed the first two examples of inducible transgene expression in pigs. The CAG-TA transgenic pigs generated in this study constitute an interesting basis for future pig models with inducible transgene expression.

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Genetically modified pigs are important models for biomedical research. For certain applications, tightly controlled transgene expression is mandatory. For instance, expression of soluble receptor activator of the NFκB ligand (RANKL) is an established principle for inducing clinically relevant osteoporosis (Mizuno et al. 2002 J. Bone Miner. Metab. 20, 337–344). However, constitutive overexpression of RANKL is lethal; thus, an inducible system is required. A second example is the expression of porcine CTLA-4Ig to prevent co-stimulation of T-cells in pig-to-primate xenotransplantation. Because constitutive expression of porcine CTLA-4Ig causes immunosuppression in pigs (Phelps et al. 2009 Xenotransplantation 16, 477–485), an inducible expression system is desirable. The Tet-On system is used most widely. It is based on a tetracycline transactivator (TA) and a transactivator response element (TARE), which drives expression of the coding sequence for the transgene product of choice. Association of doxycyclin with TA allows binding to TARE, resulting in activation of transgene expression. Although widely used in mice, this system has not yet been established in pigs. Because consistently high expression of TA is required for a functional system, we first cloned an expression vector for TA under the control of the CMV enhancer/chicken β-actin promoter (CAG). A floxed neomycin resistance cassette was linked to this vector to facilitate selection of stable clones after transfection of primary porcine kidney cells. The latter were used for nuclear transfer, and cloned embryos were transferred to synchronized recipient gilts, resulting in the birth of 5 CAG-TA transgenic piglets. A spectrum of organs and tissues was investigated by RT-PCR and Western blot for TA mRNA and protein expression. Primary kidney cells from all 5 TA transgenic piglets were transfected with TARE-RANKL and TARE-CTLA-4Ig constructs linked with a blasticidin resistance cassette to facilitate selection of double transgenic/transfected cell clones. These cell clones were individually stimulated with doxycyclin to identify cell clones with low background and high inducibility of expression of RANKL or CTLA-4Ig. For both combinations, several clones meeting these criteria were identified. Such cell clones were used for nuclear transfer to produce double-transgenic pigs. Application of doxycyclin (25 to 50 mg kg–1 of body weight/day) to a TA/TARE-RANKL transgenic pig resulted in a marked increase in plasma RANKL concentrations. In addition, 4 TA/TARE-CTLA-4Ig transgenic piglets have been born, which will undergo doxycyclin stimulation experiments in the near future. In summary, CAG-TA transgenic pigs serve as a reliable basis for inducible transgene expression, as shown in the present study for RANKL and CTLA-4Ig. Studies to show the biological functionality of the transgene are currently underway. Supported by DFG (FOR793) and BFS (FORZebRA).