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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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Differentiation of human MDSC.(a) 2D: MDSCs are seeded on a tissue culture flask at high density and allowed to differentiate. (b) AMT: MDSCs are cultured within 3D artificial muscle tissue. (c) 4F-AMT: MDSCs are cultured in 3D artificial muscle tissue and treated with 4 chemical factors. (d) 4F-AG-AMT: MDSC aggregates are cultured in 3D artificial muscle tissue and treated with 4 chemical factors. (e) Undifferentiated MDSCs on tissue culture flask. (f) 2D Differentiated MDSCs at day 14. (g) MDSCs in AMT. (h) MDSC aggregates after 4 hours of rotary suspension culture. (i) MDSC aggregates in AMT immediately after tissue construction. Scale bar indicates 250 μm.
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f2: Differentiation of human MDSC.(a) 2D: MDSCs are seeded on a tissue culture flask at high density and allowed to differentiate. (b) AMT: MDSCs are cultured within 3D artificial muscle tissue. (c) 4F-AMT: MDSCs are cultured in 3D artificial muscle tissue and treated with 4 chemical factors. (d) 4F-AG-AMT: MDSC aggregates are cultured in 3D artificial muscle tissue and treated with 4 chemical factors. (e) Undifferentiated MDSCs on tissue culture flask. (f) 2D Differentiated MDSCs at day 14. (g) MDSCs in AMT. (h) MDSC aggregates after 4 hours of rotary suspension culture. (i) MDSC aggregates in AMT immediately after tissue construction. Scale bar indicates 250 μm.

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)

Differentiation of human MDSC.(a) 2D: MDSCs are seeded on a tissue culture flask at high density and allowed to differentiate. (b) AMT: MDSCs are cultured within 3D artificial muscle tissue. (c) 4F-AMT: MDSCs are cultured in 3D artificial muscle tissue and treated with 4 chemical factors. (d) 4F-AG-AMT: MDSC aggregates are cultured in 3D artificial muscle tissue and treated with 4 chemical factors. (e) Undifferentiated MDSCs on tissue culture flask. (f) 2D Differentiated MDSCs at day 14. (g) MDSCs in AMT. (h) MDSC aggregates after 4 hours of rotary suspension culture. (i) MDSC aggregates in AMT immediately after tissue construction. Scale bar indicates 250 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Differentiation of human MDSC.(a) 2D: MDSCs are seeded on a tissue culture flask at high density and allowed to differentiate. (b) AMT: MDSCs are cultured within 3D artificial muscle tissue. (c) 4F-AMT: MDSCs are cultured in 3D artificial muscle tissue and treated with 4 chemical factors. (d) 4F-AG-AMT: MDSC aggregates are cultured in 3D artificial muscle tissue and treated with 4 chemical factors. (e) Undifferentiated MDSCs on tissue culture flask. (f) 2D Differentiated MDSCs at day 14. (g) MDSCs in AMT. (h) MDSC aggregates after 4 hours of rotary suspension culture. (i) MDSC aggregates in AMT immediately after tissue construction. Scale bar indicates 250 μm.
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