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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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Histological Analysis of Day 14 Differentiated MDSCs.(a) Low magnification image of AMT showing α-Actinin expression throughout the construct, indicating MDSCs acquire a muscle phenotype. (b) Expression of α-Actinin and TNNT2 in MDSCs differentiated under various protocols. Scale bars indicate 10 μm or 20 μm as stated in each image. Arrows point to multinucleated cells. Blue indicates nuclear DAPI staining. (c) Percentage of multinucleated cells (n > 4 nuclei). 4-factor treatment significantly reduced the percentage of multinucleated cells. ** P < 0.001.
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f4: Histological Analysis of Day 14 Differentiated MDSCs.(a) Low magnification image of AMT showing α-Actinin expression throughout the construct, indicating MDSCs acquire a muscle phenotype. (b) Expression of α-Actinin and TNNT2 in MDSCs differentiated under various protocols. Scale bars indicate 10 μm or 20 μm as stated in each image. Arrows point to multinucleated cells. Blue indicates nuclear DAPI staining. (c) Percentage of multinucleated cells (n > 4 nuclei). 4-factor treatment significantly reduced the percentage of multinucleated cells. ** P < 0.001.

Mentions: Histological assessments confirmed that MDSCs differentiate into a muscle phenotype in AMT (Figure 4a). MDSCs expressed α-actinin and TNNT2, both sarcomeric proteins, in all culture conditions (Figure 4b). However, a striated pattern was not clearly visible under 2D differentiation by either α-actinin or TNNT2 staining. MDSCs in 2D also formed elongated fibres containing many (more than 4) nuclei, which resemble skeletal myotubes (18.5 ± 2.5%, n = 18). In AMT, some cells retained the appearance of myotubes (13.1 ± 2.1%, n = 33). In AMT treated with 4 factors (4F-AMT [1.6 ± 0.6%, n = 38] and 4F-AG-AMT [2.4 ± 0.7%, n = 38]), myotubes were not readily apparent (Figure 4c). 4F-AG-AMT formed a muscle tissue with closely apposed muscle fibres that had a clear striated pattern by TNNT2 staining, which was less apparent in the 4F-AMT (no aggregate) group. These findings suggest that a combination of MDSC aggregate formation and 4 chemical factors was necessary for optimal differentiation towards a CM-like phenotype.


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)

Histological Analysis of Day 14 Differentiated MDSCs.(a) Low magnification image of AMT showing α-Actinin expression throughout the construct, indicating MDSCs acquire a muscle phenotype. (b) Expression of α-Actinin and TNNT2 in MDSCs differentiated under various protocols. Scale bars indicate 10 μm or 20 μm as stated in each image. Arrows point to multinucleated cells. Blue indicates nuclear DAPI staining. (c) Percentage of multinucleated cells (n > 4 nuclei). 4-factor treatment significantly reduced the percentage of multinucleated cells. ** P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Histological Analysis of Day 14 Differentiated MDSCs.(a) Low magnification image of AMT showing α-Actinin expression throughout the construct, indicating MDSCs acquire a muscle phenotype. (b) Expression of α-Actinin and TNNT2 in MDSCs differentiated under various protocols. Scale bars indicate 10 μm or 20 μm as stated in each image. Arrows point to multinucleated cells. Blue indicates nuclear DAPI staining. (c) Percentage of multinucleated cells (n > 4 nuclei). 4-factor treatment significantly reduced the percentage of multinucleated cells. ** P < 0.001.
Mentions: Histological assessments confirmed that MDSCs differentiate into a muscle phenotype in AMT (Figure 4a). MDSCs expressed α-actinin and TNNT2, both sarcomeric proteins, in all culture conditions (Figure 4b). However, a striated pattern was not clearly visible under 2D differentiation by either α-actinin or TNNT2 staining. MDSCs in 2D also formed elongated fibres containing many (more than 4) nuclei, which resemble skeletal myotubes (18.5 ± 2.5%, n = 18). In AMT, some cells retained the appearance of myotubes (13.1 ± 2.1%, n = 33). In AMT treated with 4 factors (4F-AMT [1.6 ± 0.6%, n = 38] and 4F-AG-AMT [2.4 ± 0.7%, n = 38]), myotubes were not readily apparent (Figure 4c). 4F-AG-AMT formed a muscle tissue with closely apposed muscle fibres that had a clear striated pattern by TNNT2 staining, which was less apparent in the 4F-AMT (no aggregate) group. These findings suggest that a combination of MDSC aggregate formation and 4 chemical factors was necessary for optimal differentiation towards a CM-like phenotype.

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