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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

Isolation of MDSCs from neonatal murine skeletal muscles. (a) Total number of nonadherent cells (per g of muscle tissue) and ratios of nonadherent cells (b) during three preplating steps (pP1–pP3). Panels (c–f) show representative images of nonadherent cells during the preplating procedure: before plating (pP0, (c)), 2 hours (pP1, (d)), 26 hours (pP2, (e)), and 74 hours (pP3, (f)) after plating. Scale: 100 μm. (g) Flow cytometric assessment of cardiac and skeletal muscle specific markers for cells from ISH0 cell population (n = 5).
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fig1: Isolation of MDSCs from neonatal murine skeletal muscles. (a) Total number of nonadherent cells (per g of muscle tissue) and ratios of nonadherent cells (b) during three preplating steps (pP1–pP3). Panels (c–f) show representative images of nonadherent cells during the preplating procedure: before plating (pP0, (c)), 2 hours (pP1, (d)), 26 hours (pP2, (e)), and 74 hours (pP3, (f)) after plating. Scale: 100 μm. (g) Flow cytometric assessment of cardiac and skeletal muscle specific markers for cells from ISH0 cell population (n = 5).

Mentions: The mechanical and enzymatic dissociation of skeletal muscles isolated from neonatal mice resulted in 27.5 ± 1.4 × 106 per gram of tissue (n = 12). Three serial preplating steps (pP1–pP3) reduced cell numbers, that is, numbers of vital nonadherent cells, to 20.3 ± 1.9 × 106 after pP1, to 9.4 ± 1.2 × 106 after pP2, and to 7.9 ± 1.0 × 106 after pP3 (Figure 1). The resulting population of nonadherent, cluster-forming cells after pP3 was termed ISH0, since it served as the initial population (day 0) of cells, which was then split and subjected to three different cell culture conditions: static incubation (I), dynamic incubation on a horizontal shaker (S), and preculture in hanging drops (H) with subsequent culture on a shaker. ISH0 cells formed clusters of spontaneously beating cells. Flow cytometric analyses (n = 5) revealed a heterogeneous cell population with a majority of cells expressing the pan-muscle marker desmin (82.5%  ± 4.4%) and substantial fractions of cells expressing cardiac troponin T (cTnT, 35.6%  ± 7.4%), stem cell lineage marker Sca-1 (32.0%  ± 3.7%), and skeletal muscle progenitor cell specific transcription factor Pax7 (19.9%  ± 10.4%). Additionally, smaller fractions of cells were also positive for the hematopoietic stem cell marker CD34 (9.0%  ± 0.8%) and cardiac transcription factor Nkx2.5 (1.9%  ± 0.1%).


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)

Isolation of MDSCs from neonatal murine skeletal muscles. (a) Total number of nonadherent cells (per g of muscle tissue) and ratios of nonadherent cells (b) during three preplating steps (pP1–pP3). Panels (c–f) show representative images of nonadherent cells during the preplating procedure: before plating (pP0, (c)), 2 hours (pP1, (d)), 26 hours (pP2, (e)), and 74 hours (pP3, (f)) after plating. Scale: 100 μm. (g) Flow cytometric assessment of cardiac and skeletal muscle specific markers for cells from ISH0 cell population (n = 5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556334&req=5

fig1: Isolation of MDSCs from neonatal murine skeletal muscles. (a) Total number of nonadherent cells (per g of muscle tissue) and ratios of nonadherent cells (b) during three preplating steps (pP1–pP3). Panels (c–f) show representative images of nonadherent cells during the preplating procedure: before plating (pP0, (c)), 2 hours (pP1, (d)), 26 hours (pP2, (e)), and 74 hours (pP3, (f)) after plating. Scale: 100 μm. (g) Flow cytometric assessment of cardiac and skeletal muscle specific markers for cells from ISH0 cell population (n = 5).
Mentions: The mechanical and enzymatic dissociation of skeletal muscles isolated from neonatal mice resulted in 27.5 ± 1.4 × 106 per gram of tissue (n = 12). Three serial preplating steps (pP1–pP3) reduced cell numbers, that is, numbers of vital nonadherent cells, to 20.3 ± 1.9 × 106 after pP1, to 9.4 ± 1.2 × 106 after pP2, and to 7.9 ± 1.0 × 106 after pP3 (Figure 1). The resulting population of nonadherent, cluster-forming cells after pP3 was termed ISH0, since it served as the initial population (day 0) of cells, which was then split and subjected to three different cell culture conditions: static incubation (I), dynamic incubation on a horizontal shaker (S), and preculture in hanging drops (H) with subsequent culture on a shaker. ISH0 cells formed clusters of spontaneously beating cells. Flow cytometric analyses (n = 5) revealed a heterogeneous cell population with a majority of cells expressing the pan-muscle marker desmin (82.5%  ± 4.4%) and substantial fractions of cells expressing cardiac troponin T (cTnT, 35.6%  ± 7.4%), stem cell lineage marker Sca-1 (32.0%  ± 3.7%), and skeletal muscle progenitor cell specific transcription factor Pax7 (19.9%  ± 10.4%). Additionally, smaller fractions of cells were also positive for the hematopoietic stem cell marker CD34 (9.0%  ± 0.8%) and cardiac transcription factor Nkx2.5 (1.9%  ± 0.1%).

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