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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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Contractile Properties of Artificial Muscle Tissue.(a) Force-strain relation of AMT. AMT increased force in response to increasing strain (Frank-Starling). * P < 0.05 vs. baseline. † P < 0.05 vs AMT. (b) Force-frequency relation of AMT at stimulation rates of 1–5 Hz. (c) Contractile response of AMT to isoproterenol. Values are expressed relative to pre-treatment values. ** P < 0.001 vs. AMT. (d) Contractile response of AMT to extracellular calcium. Calcium concentration of the bathing solution was increased from 2 mM to 5 mM.
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f5: Contractile Properties of Artificial Muscle Tissue.(a) Force-strain relation of AMT. AMT increased force in response to increasing strain (Frank-Starling). * P < 0.05 vs. baseline. † P < 0.05 vs AMT. (b) Force-frequency relation of AMT at stimulation rates of 1–5 Hz. (c) Contractile response of AMT to isoproterenol. Values are expressed relative to pre-treatment values. ** P < 0.001 vs. AMT. (d) Contractile response of AMT to extracellular calcium. Calcium concentration of the bathing solution was increased from 2 mM to 5 mM.

Mentions: All AMTs showed spontaneous beating activity from day 4, which gradually increased over the culture period and was sustained until day 14 (Supplementary Movie 1). We observed that the spontaneous beating activity of 4F-AG-AMT was visibly more vigorous than other groups. The presence of spontaneous beating activity confirms the differentiation of MDSCs into a functioning muscle phenotype. AMTs in all groups generated contractile force in response to electrical stimulation. The isometric force increased with strain (Frank-Starling mechanism) in all groups. 4F-AG-AMT (0.23 ± 0.02 mN at Lmax, n = 20) generated significantly higher force than AMT (0.16 ± 0.02 mN at Lmax, n = 16), and 4F-AMT generated less force than AMT (0.12 ± 0.02 mN at Lmax, n = 15) (Figure 5a). AMT showed a negative force-frequency relationship. In contrast, 4F-AMT and 4F-AG-AMT displayed a modestly positive force-frequency relationship at low frequencies (1–2 Hz) before becoming negative at higher stimulation frequencies (Figure 5b). 4F-AMT (134.7 ± 5.4%, n = 14, P < 0.001) and 4F-AG-AMT (124.6 ± 3.4% n = 24, P < 0.001) showed an increased positive inotropic response to isoproterenol compared to control untreated AMT (104.4 ± 2.8% n = 23, P < 0.001) (Figure 5c). All AMTs had a positive inotropic response to increased extracellular calcium, but there was no significant difference among groups (Figure 5d).


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)

Contractile Properties of Artificial Muscle Tissue.(a) Force-strain relation of AMT. AMT increased force in response to increasing strain (Frank-Starling). * P < 0.05 vs. baseline. † P < 0.05 vs AMT. (b) Force-frequency relation of AMT at stimulation rates of 1–5 Hz. (c) Contractile response of AMT to isoproterenol. Values are expressed relative to pre-treatment values. ** P < 0.001 vs. AMT. (d) Contractile response of AMT to extracellular calcium. Calcium concentration of the bathing solution was increased from 2 mM to 5 mM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Contractile Properties of Artificial Muscle Tissue.(a) Force-strain relation of AMT. AMT increased force in response to increasing strain (Frank-Starling). * P < 0.05 vs. baseline. † P < 0.05 vs AMT. (b) Force-frequency relation of AMT at stimulation rates of 1–5 Hz. (c) Contractile response of AMT to isoproterenol. Values are expressed relative to pre-treatment values. ** P < 0.001 vs. AMT. (d) Contractile response of AMT to extracellular calcium. Calcium concentration of the bathing solution was increased from 2 mM to 5 mM.
Mentions: All AMTs showed spontaneous beating activity from day 4, which gradually increased over the culture period and was sustained until day 14 (Supplementary Movie 1). We observed that the spontaneous beating activity of 4F-AG-AMT was visibly more vigorous than other groups. The presence of spontaneous beating activity confirms the differentiation of MDSCs into a functioning muscle phenotype. AMTs in all groups generated contractile force in response to electrical stimulation. The isometric force increased with strain (Frank-Starling mechanism) in all groups. 4F-AG-AMT (0.23 ± 0.02 mN at Lmax, n = 20) generated significantly higher force than AMT (0.16 ± 0.02 mN at Lmax, n = 16), and 4F-AMT generated less force than AMT (0.12 ± 0.02 mN at Lmax, n = 15) (Figure 5a). AMT showed a negative force-frequency relationship. In contrast, 4F-AMT and 4F-AG-AMT displayed a modestly positive force-frequency relationship at low frequencies (1–2 Hz) before becoming negative at higher stimulation frequencies (Figure 5b). 4F-AMT (134.7 ± 5.4%, n = 14, P < 0.001) and 4F-AG-AMT (124.6 ± 3.4% n = 24, P < 0.001) showed an increased positive inotropic response to isoproterenol compared to control untreated AMT (104.4 ± 2.8% n = 23, P < 0.001) (Figure 5c). All AMTs had a positive inotropic response to increased extracellular calcium, but there was no significant difference among groups (Figure 5d).

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