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Combined biophysical and soluble factor modulation induces cardiomyocyte differentiation from human muscle derived stem cells.

Tchao J, Han L, Lin B, Yang L, Tobita K - Sci Rep (2014)

Bottom Line: Cellular cardiomyoplasty has emerged as a novel therapy to restore contractile function of injured failing myocardium.They also express cardiac gap-junction protein, connexin-43, similar to CMs and synchronized spontaneous calcium transients.These results highlight the importance of temporal control of biophysical and soluble factors for CM differentiation from MDSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA.

ABSTRACT
Cellular cardiomyoplasty has emerged as a novel therapy to restore contractile function of injured failing myocardium. Human multipotent muscle derived stem cells (MDSC) can be a potential abundant, autologous cell source for cardiac repair. However, robust conditions for cardiomyocyte (CM) differentiation are not well established for this cell type. We have developed a new method for CM differentiation from human MDSC that combines 3-dimensional artificial muscle tissue (AMT) culture with temporally controlled biophysical cell aggregation and delivery of 4 soluble factors (microRNA-206 inhibitor, IWR-1, Lithium Chloride, and BMP-4) (4F-AG-AMT). The 4F-AG-AMT displayed cardiac-like response to β-adrenergic stimulation and contractile properties. 4F-AG-AMT expressed major cardiac (NKX2-5, GATA4, TBX5, MEF2C) transcription factors and structural proteins. They also express cardiac gap-junction protein, connexin-43, similar to CMs and synchronized spontaneous calcium transients. These results highlight the importance of temporal control of biophysical and soluble factors for CM differentiation from MDSCs.

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Intercellular Coupling of MDSCs.(a) Intracellular calcium transients of spontaneously beating MDSC-CMs recorded using Rhod-2 AM dye from 4 different locations. Trace shows that the transients begin and end in a synchronous manner (vertical lines). (b) Co-staining of 4-factor treated day 14 4F-AG-AMT with GJA1 (red), α-Actinin (green), and DAPI (blue). Arrows show gap junction formation between cells. Scale indicates 10 μm. (c) Co-staining of day 14 2D Differentiated MSDCs with GJA1 (red), α-Actinin (green), and DAPI (blue). Scale bar indicates 10 μm.
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f6: Intercellular Coupling of MDSCs.(a) Intracellular calcium transients of spontaneously beating MDSC-CMs recorded using Rhod-2 AM dye from 4 different locations. Trace shows that the transients begin and end in a synchronous manner (vertical lines). (b) Co-staining of 4-factor treated day 14 4F-AG-AMT with GJA1 (red), α-Actinin (green), and DAPI (blue). Arrows show gap junction formation between cells. Scale indicates 10 μm. (c) Co-staining of day 14 2D Differentiated MSDCs with GJA1 (red), α-Actinin (green), and DAPI (blue). Scale bar indicates 10 μm.

Mentions: We assessed functional intercellular coupling by measuring intracellular calcium transients in AMT during spontaneous beating. AMT treated with 4 factors (4F-AMT and 4F-AG-AMT) showed that calcium transients in each cell were synchronous over the entire field of view (Figure 6a, Supplementary Movie 2). We co-stained cells for GJA1 and α-Actinin to identify gap junction intercellular coupling between differentiated myocytes. We observed a distinct dot-like pattern of connexin-43 staining in cells treated with 4 factors, which included concentrated staining at the border between adjacent cells (Figure 6b, yellow arrows). In contrast, cells that were differentiated under the 2D protocol stained positive for GJA1 and α-Actinin, but a distinct staining pattern was not observed (Figure 6c). Taken together, these data suggest that treatment of AMT with chemical factors can improve intercellular coupling.


Combined biophysical and soluble factor modulation induces cardiomyocyte differentiation from human muscle derived stem cells.

Tchao J, Han L, Lin B, Yang L, Tobita K - Sci Rep (2014)

Intercellular Coupling of MDSCs.(a) Intracellular calcium transients of spontaneously beating MDSC-CMs recorded using Rhod-2 AM dye from 4 different locations. Trace shows that the transients begin and end in a synchronous manner (vertical lines). (b) Co-staining of 4-factor treated day 14 4F-AG-AMT with GJA1 (red), α-Actinin (green), and DAPI (blue). Arrows show gap junction formation between cells. Scale indicates 10 μm. (c) Co-staining of day 14 2D Differentiated MSDCs with GJA1 (red), α-Actinin (green), and DAPI (blue). Scale bar indicates 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196107&req=5

f6: Intercellular Coupling of MDSCs.(a) Intracellular calcium transients of spontaneously beating MDSC-CMs recorded using Rhod-2 AM dye from 4 different locations. Trace shows that the transients begin and end in a synchronous manner (vertical lines). (b) Co-staining of 4-factor treated day 14 4F-AG-AMT with GJA1 (red), α-Actinin (green), and DAPI (blue). Arrows show gap junction formation between cells. Scale indicates 10 μm. (c) Co-staining of day 14 2D Differentiated MSDCs with GJA1 (red), α-Actinin (green), and DAPI (blue). Scale bar indicates 10 μm.
Mentions: We assessed functional intercellular coupling by measuring intracellular calcium transients in AMT during spontaneous beating. AMT treated with 4 factors (4F-AMT and 4F-AG-AMT) showed that calcium transients in each cell were synchronous over the entire field of view (Figure 6a, Supplementary Movie 2). We co-stained cells for GJA1 and α-Actinin to identify gap junction intercellular coupling between differentiated myocytes. We observed a distinct dot-like pattern of connexin-43 staining in cells treated with 4 factors, which included concentrated staining at the border between adjacent cells (Figure 6b, yellow arrows). In contrast, cells that were differentiated under the 2D protocol stained positive for GJA1 and α-Actinin, but a distinct staining pattern was not observed (Figure 6c). Taken together, these data suggest that treatment of AMT with chemical factors can improve intercellular coupling.

Bottom Line: Cellular cardiomyoplasty has emerged as a novel therapy to restore contractile function of injured failing myocardium.They also express cardiac gap-junction protein, connexin-43, similar to CMs and synchronized spontaneous calcium transients.These results highlight the importance of temporal control of biophysical and soluble factors for CM differentiation from MDSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA.

ABSTRACT
Cellular cardiomyoplasty has emerged as a novel therapy to restore contractile function of injured failing myocardium. Human multipotent muscle derived stem cells (MDSC) can be a potential abundant, autologous cell source for cardiac repair. However, robust conditions for cardiomyocyte (CM) differentiation are not well established for this cell type. We have developed a new method for CM differentiation from human MDSC that combines 3-dimensional artificial muscle tissue (AMT) culture with temporally controlled biophysical cell aggregation and delivery of 4 soluble factors (microRNA-206 inhibitor, IWR-1, Lithium Chloride, and BMP-4) (4F-AG-AMT). The 4F-AG-AMT displayed cardiac-like response to β-adrenergic stimulation and contractile properties. 4F-AG-AMT expressed major cardiac (NKX2-5, GATA4, TBX5, MEF2C) transcription factors and structural proteins. They also express cardiac gap-junction protein, connexin-43, similar to CMs and synchronized spontaneous calcium transients. These results highlight the importance of temporal control of biophysical and soluble factors for CM differentiation from MDSCs.

Show MeSH