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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

Expression of cardiomyocyte-specific transcripts in MDSCs cultured under dynamic support. Skeletal myoblasts, purified embryonic stem cell-derived cardiomyocytes, and cells from I, S, and H cell culture conditions on days 4 and 12 of cultivation were analyzed by qPCR. Expression levels of indicated genes were normalized to the reference gene β-actin and displayed as relative expression compared to ISH0 cells. Calculation of statistical significance was based on ΔCt values (see Figure S8). ∗/∗∗/∗∗∗p < 0.05/0.01/0.001.
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fig4: Expression of cardiomyocyte-specific transcripts in MDSCs cultured under dynamic support. Skeletal myoblasts, purified embryonic stem cell-derived cardiomyocytes, and cells from I, S, and H cell culture conditions on days 4 and 12 of cultivation were analyzed by qPCR. Expression levels of indicated genes were normalized to the reference gene β-actin and displayed as relative expression compared to ISH0 cells. Calculation of statistical significance was based on ΔCt values (see Figure S8). ∗/∗∗/∗∗∗p < 0.05/0.01/0.001.

Mentions: The expression of connexin 43 (Cx43), α-myosin heavy chain 6 (MYH6), cTnT, ACTN2, and Nkx2.5 were analyzed by qPCR on the transcript level. Purified ESC derived CMs and MBs were used as controls. Compared to CMs and MBs, ISH0 showed an intermediate expression level for MYH6, cTnT, and Nkx2.5, while the expression level for Cx43 of ISH0 was similar to MBs (Figures 4 and S8). Comparing cells from day 4 and day 12 of the three different culture conditions to ISH0 cells, the expressions of MYH6 and cTnT were already increased more than 5-fold in cells at day 4 (I4 and S4 versus ISH0: p < 0.05; H4 versus ISH0: p < 0.001) and further increased in I12 (13.6-fold; p < 0.01 versus ISH0) and H12 cells (55.2-fold; p < 0.001 versus ISH0). The expression levels of cTnT were more than 3-fold higher in cells at day 4 in all cell culture conditions compared to ISH0 (I4 and H4 versus ISH0: p < 0.01; S4 versus ISH0: p < 0.05). The highest level of cTnT expression was detected in H12 cells (17.5-fold; p < 0.001 versus ISH0). During 12-day cultivation in all three culture conditions, Cx43 and ACTN2 showed only minor changes in expression levels, ranging from 0.5- to 2-fold (Figures 4 and S8). Nkx2.5 showed a tendency for increased expression, especially for condition H (H4: 27x, H12: 182x), but without reaching significance, presumably, because of very low overall expression levels (1,000-fold less than endogenous control).


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)

Expression of cardiomyocyte-specific transcripts in MDSCs cultured under dynamic support. Skeletal myoblasts, purified embryonic stem cell-derived cardiomyocytes, and cells from I, S, and H cell culture conditions on days 4 and 12 of cultivation were analyzed by qPCR. Expression levels of indicated genes were normalized to the reference gene β-actin and displayed as relative expression compared to ISH0 cells. Calculation of statistical significance was based on ΔCt values (see Figure S8). ∗/∗∗/∗∗∗p < 0.05/0.01/0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556334&req=5

fig4: Expression of cardiomyocyte-specific transcripts in MDSCs cultured under dynamic support. Skeletal myoblasts, purified embryonic stem cell-derived cardiomyocytes, and cells from I, S, and H cell culture conditions on days 4 and 12 of cultivation were analyzed by qPCR. Expression levels of indicated genes were normalized to the reference gene β-actin and displayed as relative expression compared to ISH0 cells. Calculation of statistical significance was based on ΔCt values (see Figure S8). ∗/∗∗/∗∗∗p < 0.05/0.01/0.001.
Mentions: The expression of connexin 43 (Cx43), α-myosin heavy chain 6 (MYH6), cTnT, ACTN2, and Nkx2.5 were analyzed by qPCR on the transcript level. Purified ESC derived CMs and MBs were used as controls. Compared to CMs and MBs, ISH0 showed an intermediate expression level for MYH6, cTnT, and Nkx2.5, while the expression level for Cx43 of ISH0 was similar to MBs (Figures 4 and S8). Comparing cells from day 4 and day 12 of the three different culture conditions to ISH0 cells, the expressions of MYH6 and cTnT were already increased more than 5-fold in cells at day 4 (I4 and S4 versus ISH0: p < 0.05; H4 versus ISH0: p < 0.001) and further increased in I12 (13.6-fold; p < 0.01 versus ISH0) and H12 cells (55.2-fold; p < 0.001 versus ISH0). The expression levels of cTnT were more than 3-fold higher in cells at day 4 in all cell culture conditions compared to ISH0 (I4 and H4 versus ISH0: p < 0.01; S4 versus ISH0: p < 0.05). The highest level of cTnT expression was detected in H12 cells (17.5-fold; p < 0.001 versus ISH0). During 12-day cultivation in all three culture conditions, Cx43 and ACTN2 showed only minor changes in expression levels, ranging from 0.5- to 2-fold (Figures 4 and S8). Nkx2.5 showed a tendency for increased expression, especially for condition H (H4: 27x, H12: 182x), but without reaching significance, presumably, because of very low overall expression levels (1,000-fold less than endogenous control).

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