A three-dimensional gel bioreactor for assessment of cardiomyocyte induction in skeletal muscle-derived stem cells

Abstract

Skeletal muscle-derived stem cells (MDSCs) are able to differentiate into cardiomyocytes (CMs). However, it remains to be investigated whether differentiated CMs contract similar to native CMs. Here, we developed a three-dimensional collagen gel bioreactor (3DGB) that induces a working CM phenotype from MDSCs, and the contractile properties are directly measured as an engineered cardiac tissue. Neonate rat MDSCs were isolated from hind-leg muscles via the preplate technique. Isolated MDSCs were approximately 60% positive to Sca-1 and negative to CD34, CD45, or c-kit antigens. We sorted Sca-1(-) MDSCs and constructed MDSC-3DGBs by mixing MDSCs with acid soluble rat tail collagen type-I and matrix factors. MDSC-3DGB exhibited spontaneous cyclic contraction by culture day 7. MDSC-3DGB expressed cardiac-specific genes and proteins. Histological assessment revealed that cardiac-specific troponin-T and -I expressed in a typical striation pattern and connexin-43 was expressed similar to the native fetal ventricular papillary muscle. beta-Adrenergic stimulation increased MDSC-3DGB spontaneous beat frequency. MDSC-3DGB generated contractile force and intracellular calcium ion transients similar to engineered cardiac tissue from native cardiac cells. Results suggest that MDSC-3DGB induces a working CM phenotype in MDSCs and is a useful 3D culture system to directly assess the contractile properties of differentiated CMs in vitro.

FIG. 1.

Histologic analysis of MDSC-3D collagen gel bioreactor (3DGB). (A) Phase contract image of culture day 7 MDSC-3DGB. Scale bar indicates 500 μm. Red box indicates the area of the tissue where high-magnification images are taken. (B) α-Sarcomeric actinin expression in MDSC-3DGB. Scale bar indicates 500 μm. (C) Cardiac troponin-T (cTn-T) expressed in oriented cells of MDSC-3DGB. Blue staining (DAPI) indicates nuclei. Scale bar indicates 50 μm. The white double-head arrow indicates MDSC-3DGB longitudinal axis. (D) cTn-T expression of MDSC-2D. Scale bar indicates 20 μm. (E) cTn-T expression of MDSC-3DGB was a typical striated muscle pattern at a higher magnification. Scale bar indicates 20 μm. (F) Cardiac troponin-I (cTn-I) also expressed in a typical striated pattern (white arrowheads). Scale bar indicates 20 μm. 2D, two-dimensional; MDSC, muscle-derived stem cell; DAPI, 4′,6-diamidino-2-phenylindole. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Native fetal left ventricular papillary muscle at gestational day 20 stained for (A) α-sarcomeric actinin (green). (B) Gap junction protein Cx-43 (red), and (C) merged. Culture day 7 MDSC-3DGB stained for (D) α-sarcomeric actinin (green). (E) Gap junction protein Cx-43 (red), and (F) merged. Cx-43 expression of MDSC-3DGB was similar to gestational day 20 fetal left ventricular papillary muscle Cx-43 expression. Blue staining (DAPI) indicates nuclei. Scale bar indicates 10 μm. Cx-43, connexin-43. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Cardiac-specific mRNA expression. Lane 1, 2D-MDSC evaluated after 7 days in culture; lane 2, MDSC-aggregate evaluated after 24 h rotation culture; lane 3, 3D-MDSC (without MDSC-aggregate formation) evaluated 7 days after tissue construction; lane 4, MDSC-3DGB evaluated 7 days after tissue construction; lane 5, adult rat ventricular tissue.

FIG. 4.

Cardiac-specific protein quantification. (A) Representative Western blot analysis of lane 1, MDSC-aggregate evaluated after 24 h rotation culture; lane 2, 3D-MDSC (without MDSC-aggregate formation) evaluated 7 days after tissue formation; lane 3, MDSC-3DGB evaluated 7 days after tissue formation; lane 4, gestational day 20 fetal rat ventricular tissue; lane 5, twelve-week-old adult rat ventricular tissue; lane 6, gestational day 20 fetal rat lower leg skeletal muscle; lane 7, twelve-week-old adult rat gastrocnemius muscle. (B) Densitometric data normalized to β-actin expression (average expression vs. β-actin, %). *p < 0.05 versus MDSC-aggregate culture normalized expression. ^†p < 0.05 versus 3D-MDSC culture normalized expression. ^‡p < 0.05 versus MDSC-3DGB culture normalized expression. Each lane contains 20 μg of protein per sample, and experiments were repeated in triplicate.

FIG. 5.

Effects of ISP and CdCl₂ treatment on spontaneous beating activity of MDSC-3DGB. Culture day 7 MDSC-3DGBs treated with 2 μM ISP for 5 min increased spontaneous beat frequency. MDSC-3DGBs treated with 1 mM CdCl₂ for 5 min decreased spontaneous beating. *p < 0.05 versus pretreatment. ISP, isoproterenol; CdCl₂, cadmium chloride.

FIG. 6.

Biomechanical testing of MDSC-3DGB. (A) MDSC-3DGB mounted on a mechanical testing station (white arrow). Scribed x-axis minor scale divisions represent 1 mm. (B) Representative contractile force tracing of MDSC-3DGB at increasing resting lengths. (C) Active force–strain relations of culture day 7 MDSC-3DGB, culture day 7 3D-MDSC, and EFCT at strain deviations of 0 to 0.15. ^†p < 0.05 versus MDSC-3DGB. Active force increased in response to increased strain (positive Frank-Starling response, *p < 0.05, analysis of variance). Color images available online at www.liebertonline.com/ten.

FIG. 7.

Simultaneous contractile force and [Ca²⁺]_i transient measurement of MDSC-3DGB. (A) Representative single beat force (red) fluorescence at 340 nm (green) and at 380 nm (blue), and the relative fluorescence (ratio [R = F₃₄₀/F₃₈₀]) (black) tracing of culture day 7 MDSC-3DGB electrically stimulated at 1 Hz at 50 V and 4 ms duration. A rise in [Ca²⁺]_i preceded force generation and each [Ca²⁺]_i transient was associated with a concurrent contraction. (B) Culture day 7 MDSC-3DGB electrically stimulated at rates of 1 to 6 Hz at 50 V and 4 ms duration. MDSC-3DGB showed increased diastolic [Ca²⁺]_i and reduced systolic [Ca²⁺]_i transient ratios (black) associated with increased diastolic and decreased active force (red) at increasing pacing rates, similar to immature myocardium. [Ca²⁺]_i, intracellular free calcium ion. Color images available online at www.liebertonline.com/ten.