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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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(a) Gene Expression of Day 14 Differentiated MDSCs. Muscle-specific gene expression of MDSCs is dependent on culture conditions. Values are expressed as fold change in gene expression and normalized to 2D differentiation protocol (2D). Red line indicates twofold change. *P < 0.05. **P < 0.001. (b) NKX2-5 protein expression in 4F-AG-AMT and AMT was detected by immunohistochemistry.
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f3: (a) Gene Expression of Day 14 Differentiated MDSCs. Muscle-specific gene expression of MDSCs is dependent on culture conditions. Values are expressed as fold change in gene expression and normalized to 2D differentiation protocol (2D). Red line indicates twofold change. *P < 0.05. **P < 0.001. (b) NKX2-5 protein expression in 4F-AG-AMT and AMT was detected by immunohistochemistry.

Mentions: We examined gene expression of MDSCs differentiated under the 4 different protocols (Figure 2) of combined biophysical and soluble factors (Figure 3a). All genes were expressed regardless of the protocol used. 4F-AMT increased expression of cardiac transcription factors NKX2-5, GATA4, and to a lesser extent, TBX5 (n = 6). Aggregate formation combined with 4 factor treatment (4F-AG-AMT) increased MEF2C, MYH6, and MYH7 expression in addition to the aforementioned genes (n = 6). Interestingly, MYOD1 also increased under the 4F-AMT and 4F-AG-AMT protocols (n = 6). MYOG was also elevated in the 4F-AMT protocol (n = 6). GJA1 expression remained unchanged across the differentiation protocols (n = 6). These data indicate that human MDSCs express both cardiac and skeletal muscle genes, a characteristic shared with iPS cell derived cardiomyocytes and immature developing cardiac and skeletal muscle12. 4F-AG-AMT also expressed NKX2-5 protein by immunohistochemistry, while AMT culture alone induced limited expression with many cells remaining negative for NKX2-5 (Figure 3b).


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)

(a) Gene Expression of Day 14 Differentiated MDSCs. Muscle-specific gene expression of MDSCs is dependent on culture conditions. Values are expressed as fold change in gene expression and normalized to 2D differentiation protocol (2D). Red line indicates twofold change. *P < 0.05. **P < 0.001. (b) NKX2-5 protein expression in 4F-AG-AMT and AMT was detected by immunohistochemistry.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: (a) Gene Expression of Day 14 Differentiated MDSCs. Muscle-specific gene expression of MDSCs is dependent on culture conditions. Values are expressed as fold change in gene expression and normalized to 2D differentiation protocol (2D). Red line indicates twofold change. *P < 0.05. **P < 0.001. (b) NKX2-5 protein expression in 4F-AG-AMT and AMT was detected by immunohistochemistry.
Mentions: We examined gene expression of MDSCs differentiated under the 4 different protocols (Figure 2) of combined biophysical and soluble factors (Figure 3a). All genes were expressed regardless of the protocol used. 4F-AMT increased expression of cardiac transcription factors NKX2-5, GATA4, and to a lesser extent, TBX5 (n = 6). Aggregate formation combined with 4 factor treatment (4F-AG-AMT) increased MEF2C, MYH6, and MYH7 expression in addition to the aforementioned genes (n = 6). Interestingly, MYOD1 also increased under the 4F-AMT and 4F-AG-AMT protocols (n = 6). MYOG was also elevated in the 4F-AMT protocol (n = 6). GJA1 expression remained unchanged across the differentiation protocols (n = 6). These data indicate that human MDSCs express both cardiac and skeletal muscle genes, a characteristic shared with iPS cell derived cardiomyocytes and immature developing cardiac and skeletal muscle12. 4F-AG-AMT also expressed NKX2-5 protein by immunohistochemistry, while AMT culture alone induced limited expression with many cells remaining negative for NKX2-5 (Figure 3b).

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
Related in: MedlinePlus