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      Development of Functional I f Channels in mMSCs after Transfection with mHCN4: Effects on Cell Morphology and Mechanical Activity in vitro

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          Objective: To study the functional properties of I<sub>f</sub> channels and the changes in mechanical activity of mouse mesenchymal stem cells (mMSCs) transfected with mHCN4. Methods: mMSCs were purified by using CD11b-immunomagnetic microbeads and transfected with pMSCV-mHCN4-EGFP or pMSCV-EGFP. We examined the kinetic characteristics of the mHCN4 channel. The morphological changes of positively transfected mMSCs were investigated at the same time. Results: The I<sub>f</sub> current recorded from the experimental group was sensitive to extracellular Cs<sup>+</sup> (–44.5 ± 4.2 vs. –5.5 ± 1.0 pA/pF, p < 0.001). The half-maximal activation was –99.0 ± 5.8 mV. The time constant of activation was 451 ± 61 ms under –140 mV. The control cells did not show the current under the same conditions. The absolute values of half-maximal activation decreased in the presence of cAMP or cGMP in the experimental group (–78.6 ± 10.4 and –85.7 ± 8.6 vs. –99.0 ± 5.8 mV, respectively, p < 0.05). mMSCs transfected with pMSCV-mHCN4-EGFP could form spontaneous beating cells. Extracellular Cs<sup>+</sup> decreased the beating rate significantly (196 ± 50 vs. 66 ± 23 bmp, p < 0.01). Conclusions: Functional I<sub>f</sub> channels can be reconstructed in mMSCs infected with mHCN4. mMSCs modified by successful transfection with mHCN4 can differentiate so as to develop spontaneous mechanical activity.

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            Molecular diversity of pacemaker ion channels.

            Ionic currents activated by hyperpolarization and regulated by cyclic nucleotides were first discovered more than 20 years ago. Recently the molecular identity of the underlying channels has been unveiled. The structural features of the protein sequences are discussed and related to the mechanisms of activation, selectivity for cyclic nucleotides, and ion permeation. Coverage includes a comparison of the biophysical properties of recombinant and native channels and their significance for the physiological functions of these channels.
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              In vivo contribution of murine mesenchymal stem cells into multiple cell-types under minimal damage conditions.

              Murine mesenchymal stem cells are capable of differentiating in vitro into different lineages under stimulation with certain cytokines, growth factors and chemicals. However, the true capacity of these cells to contribute to different cell-types in vivo is still unclear, especially under minimal injury conditions. In this study, we describe a method of purifying murine mesenchymal stem cells from bone marrow and efficiently transducing them using a lentivirus vector expressing the eGFP reporter gene. Lentivirus-transduced mesenchymal stem cells retained their in vitro ability to differentiate into adipocytes, osteocytes and chondrocytes as well as into myocyte- and astrocyte-like cells. eGFP-mesenchymal stem cells were delivered systemically into minimally injured syngeneic mice. Tracking and tissue-specific differentiation were determined by PCR and immunohistochemistry, respectively. We found donor-derived hepatocytes, lung epithelial cells, myofibroblasts, myofibers and renal tubular cells in some of the recipient mice. Our data indicate that even in the absence of substantial injury, phenotypically defined murine mesenchymal stem cells could acquire tissue specific morphology and antigen expression and thus contribute to different tissue cell-types in vivo.

                Author and article information

                S. Karger AG
                December 2008
                01 July 2008
                : 112
                : 2
                : 114-121
                aDepartment of Cardiology, Southwest Hospital, bInstitute of Immunology, PLA, and cDepartment of Pharmacology, Third Military Medical University, Chongqing, China
                141919 Cardiology 2009;112:114–121
                © 2008 S. Karger AG, Basel

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                Figures: 8, References: 29, Pages: 8
                Original Research


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