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Dynamic Support Culture of Murine Skeletal Muscle-Derived Stem Cells Improves Their Cardiogenic Potential In Vitro.

Neef K, Treskes P, Xu G, Drey F, Srinivasan SP, Saric T, Nembo E, Semmler J, Nguemo F, Stamm C, Cowan DB, Deppe AC, Scherner M, Wittwer T, Hescheler J, Wahlers T, Choi YH - Stem Cells Int (2015)

Bottom Line: A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation.Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli.Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic Surgery, Heart Center, University of Cologne, 50937 Cologne, Germany ; Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany.

ABSTRACT
Ischemic heart disease is the main cause of death in western countries and its burden is increasing worldwide. It typically involves irreversible degeneration and loss of myocardial tissue leading to poor prognosis and fatal outcome. Autologous cells with the potential to regenerate damaged heart tissue would be an ideal source for cell therapeutic approaches. Here, we compared different methods of conditional culture for increasing the yield and cardiogenic potential of murine skeletal muscle-derived stem cells. A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation. In contrast to static culture conditions, dynamic culture with or without previous hanging drop preculture led to significantly increased cluster diameters and the expression of cardiac specific markers on the protein and mRNA level. Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli. This data indicates that skeletal muscle-derived stem cells are capable of adopting enhanced cardiac muscle cell-like properties by applying specific culture conditions. Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells.

No MeSH data available.


Related in: MedlinePlus

Electrophysiological analyses of MDSC-derived cells. (a) Representative action potential traces of initial cell population (ISH0) and after applying different cell culture conditions for 12 days (I12, S12, and H12) as measured by a whole-cell patch-clamp in current-clamp mode. (b) Voltage clamp measurements for recording of the pacemaker current If. The applied voltage protocol is shown above the traces, which are representative for cells which do not express (upper trace) and which express small (lower trace) If currents.
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fig5: Electrophysiological analyses of MDSC-derived cells. (a) Representative action potential traces of initial cell population (ISH0) and after applying different cell culture conditions for 12 days (I12, S12, and H12) as measured by a whole-cell patch-clamp in current-clamp mode. (b) Voltage clamp measurements for recording of the pacemaker current If. The applied voltage protocol is shown above the traces, which are representative for cells which do not express (upper trace) and which express small (lower trace) If currents.

Mentions: The frequency of spontaneous action potentials (APs) was similar in ISH0 (360.6 ± 81.6 beats/min) and H12 cells (444.4 ± 39.3 beats/min), while cells from I12 and S12 showed more than 2x higher frequencies (Table 1). Analysis of AP parameters revealed that I12 cells showed a more depolarized maximum diastolic potential (MDP), shorter AP duration (APD), and lower maximum AP upstroke velocity (Vmax) than ISH0, S12, and H12 cells (Figure 5(b)). However, the morphology of APs from cells cultured under different conditions was similar, characterized by a slow depolarization before each AP, fast upstroke, and short AP duration, without a plateau phase after the AP upstroke (Figure 5(a)). Further, APs (Figure 5(b), left panel) and If currents (Figure 5(b), right panel) were recorded from the same cell by switching from current-clamping to voltage-clamping as previously described [27]. We examined the functional expression of If in spontaneous beating cells generated under ISH10, I12, S12, and H12. The typical representative If current traces (Figure 5(b), right) recorded on nonbeating (upper) and beating (lower) ISH10 cells are depicted. Several beating cells revealed the presence of If current whereas most of the nonbeating cells were characterized by the absence of If current, confirming the important role of If in the generation and modulation of spontaneous beating activity of the cells.


Dynamic Support Culture of Murine Skeletal Muscle-Derived Stem Cells Improves Their Cardiogenic Potential In Vitro.

Neef K, Treskes P, Xu G, Drey F, Srinivasan SP, Saric T, Nembo E, Semmler J, Nguemo F, Stamm C, Cowan DB, Deppe AC, Scherner M, Wittwer T, Hescheler J, Wahlers T, Choi YH - Stem Cells Int (2015)

Electrophysiological analyses of MDSC-derived cells. (a) Representative action potential traces of initial cell population (ISH0) and after applying different cell culture conditions for 12 days (I12, S12, and H12) as measured by a whole-cell patch-clamp in current-clamp mode. (b) Voltage clamp measurements for recording of the pacemaker current If. The applied voltage protocol is shown above the traces, which are representative for cells which do not express (upper trace) and which express small (lower trace) If currents.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556334&req=5

fig5: Electrophysiological analyses of MDSC-derived cells. (a) Representative action potential traces of initial cell population (ISH0) and after applying different cell culture conditions for 12 days (I12, S12, and H12) as measured by a whole-cell patch-clamp in current-clamp mode. (b) Voltage clamp measurements for recording of the pacemaker current If. The applied voltage protocol is shown above the traces, which are representative for cells which do not express (upper trace) and which express small (lower trace) If currents.
Mentions: The frequency of spontaneous action potentials (APs) was similar in ISH0 (360.6 ± 81.6 beats/min) and H12 cells (444.4 ± 39.3 beats/min), while cells from I12 and S12 showed more than 2x higher frequencies (Table 1). Analysis of AP parameters revealed that I12 cells showed a more depolarized maximum diastolic potential (MDP), shorter AP duration (APD), and lower maximum AP upstroke velocity (Vmax) than ISH0, S12, and H12 cells (Figure 5(b)). However, the morphology of APs from cells cultured under different conditions was similar, characterized by a slow depolarization before each AP, fast upstroke, and short AP duration, without a plateau phase after the AP upstroke (Figure 5(a)). Further, APs (Figure 5(b), left panel) and If currents (Figure 5(b), right panel) were recorded from the same cell by switching from current-clamping to voltage-clamping as previously described [27]. We examined the functional expression of If in spontaneous beating cells generated under ISH10, I12, S12, and H12. The typical representative If current traces (Figure 5(b), right) recorded on nonbeating (upper) and beating (lower) ISH10 cells are depicted. Several beating cells revealed the presence of If current whereas most of the nonbeating cells were characterized by the absence of If current, confirming the important role of If in the generation and modulation of spontaneous beating activity of the cells.

Bottom Line: A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation.Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli.Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiothoracic Surgery, Heart Center, University of Cologne, 50937 Cologne, Germany ; Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany.

ABSTRACT
Ischemic heart disease is the main cause of death in western countries and its burden is increasing worldwide. It typically involves irreversible degeneration and loss of myocardial tissue leading to poor prognosis and fatal outcome. Autologous cells with the potential to regenerate damaged heart tissue would be an ideal source for cell therapeutic approaches. Here, we compared different methods of conditional culture for increasing the yield and cardiogenic potential of murine skeletal muscle-derived stem cells. A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation. In contrast to static culture conditions, dynamic culture with or without previous hanging drop preculture led to significantly increased cluster diameters and the expression of cardiac specific markers on the protein and mRNA level. Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli. This data indicates that skeletal muscle-derived stem cells are capable of adopting enhanced cardiac muscle cell-like properties by applying specific culture conditions. Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells.

No MeSH data available.


Related in: MedlinePlus