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. 2012 Jul;18(13-14):1322-33.
doi: 10.1089/ten.TEA.2011.0278. Epub 2012 Jun 25.

Myocyte-depleted engineered cardiac tissues support therapeutic potential of mesenchymal stem cells

Gregory W Serrao  1 , Irene C Turnbull, Damian Ancukiewicz, Do Eun Kim, Evan Kao, Timothy J Cashman, Lahouaria Hadri, Roger J Hajjar, Kevin D Costa
Affiliations

Myocyte-depleted engineered cardiac tissues support therapeutic potential of mesenchymal stem cells

Gregory W Serrao et al. Tissue Eng Part A. 2012 Jul.

Abstract

The therapeutic potential of mesenchymal stem cells (MSCs) for restoring cardiac function after cardiomyocyte loss remains controversial. Engineered cardiac tissues (ECTs) offer a simplified three-dimensional in vitro model system to evaluate stem cell therapies. We hypothesized that contractile properties of dysfunctional ECTs would be enhanced by MSC treatment. ECTs were created from neonatal rat cardiomyocytes with and without bone marrow-derived adult rat MSCs in a type-I collagen and Matrigel scaffold using custom elastomer molds with integrated cantilever force sensors. Three experimental groups included the following: (1) baseline condition ECT consisting only of myocytes, (2) 50% myocyte-depleted ECT, modeling a dysfunctional state, and (3) 50% myocyte-depleted ECT plus 10% MSC, modeling dysfunctional myocardium with intervention. Developed stress (DS) and pacing threshold voltage (VT) were measured using 2-Hz field stimulation at 37°C on culture days 5, 10, 15, and 20. By day 5, DS of myocyte-depleted ECTs was significantly lower than baseline, and VT was elevated. In MSC-supplemented ECTs, DS and VT were significantly better than myocyte-depleted values, approaching baseline ECTs. Findings were similar through culture day 15, but lost significance at day 20. Trends in DS were partly explained by changes in the cell number and alignment with time. Thus, supplementing myocyte-depleted ECTs with MSCs transiently improved contractile function and compensated for a 50% loss of cardiomyocytes, mimicking recent animal studies and clinical trials and supporting the potential of MSCs for myocardial therapy.

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Figures

FIG. 1.
FIG. 1.
Engineered cardiac tissue (ECT) creation. (A) Diagram of cell–matrix mixture pipetted into a polydimethylsiloxane (PDMS) mold; gel formation initiates within first 2 h, leading to compaction and self-assembly of cylindrical ECTs anchored by end posts. (B) Image of a custom PDMS mold with removable inserts. Top panel: PDMS mold with inserts in place creating a defined 2×2×13-mm-rectangular well with posts near each end. Scale bar=10 mm. Lower panel: PDMS mold disassembled, with inserts on the side. (C) Image of ECT. Top panel: Top view of ECT attached to end posts. Scale bar=1 mm. Lower panel: Side view of ECT while contracting and inducing inward deflection of the posts.
FIG. 2.
FIG. 2.
Analysis of the post deflection based on the elastic beam theory, allowing calculation of the tissue force applied at some distance from the tip of the post where deflection is measured. (A) Schematic of the flexible post analysis following the elastic beam bending equation. F is tissue contraction force; E, R, L represent Young's modulus, radius, and length of the PDMS posts, respectively; a is the height of the tissue on the post; δ is measured tip deflection. (B) Representative tracing of ECT contraction during field stimulation at 2 Hz. Each spike represents post deflection, δ, during a tissue twitch. (C) Detected beating frequency of representative ECT (using fast Fourier transform analysis of post deflection) versus prescribed pacing frequency. The dotted line indicates a slope of unity, verifying measured beating frequency is equivalent to prescribed pacing frequency until the loss of capture at 14 Hz. (D) Corresponding force–frequency relationship shows a decreasing trend over the capture range from 1 to 13 Hz.
FIG. 3.
FIG. 3.
Double-labeling immunofluorescence of representative sections for three ECT groups at culture day 20: (A) baseline ECT composed of 15 M cells/mL, (B) myocyte-depleted ECT with half the amount of cells (7.5 M cells/mL), and (C) myocyte-depleted ECT (7.5 M cells/mL) supplemented with mesenchymal stem cell (MSC; 0.75 M cells/mL). Grayscale confocal images reveal sarcomeres in α-actinin-expressing cells, and a punctate pattern of connexin 43 (Cx43) expression. In merged images, Cx43 (green) often localizes along the boundaries of α-actinin-positive (red) cells. Nuclei are counterstained with 4′,6 diamidino-2-phenylindole (DAPI; blue). Scale bar=25 μm. Color images available online at www.liebertpub.com/tea
FIG. 4.
FIG. 4.
Cell number and distribution within ECT. (A) Hematoxylin and eosin stain of representative sections from each group at day 20 shows good tissue integrity with cells distributed throughout the ECT. Scale bar=50 μm. (B) Confocal microscope images of DAPI-stained nuclei in full-width ECT sections at culture day 20. Scale bar=100 μm. (C) Cell number determined by counting DAPI-stained nuclei in full-width images (as in panel B) of one representative ECT per group at each time point. Mean values and standard deviations describe data from three locations (left, middle, and right) along each tissue sample. (D) Distribution of cell data presented in panel C. Three bars for each tissue group indicate the number of nuclei in three adjacent zones (edge, center, and edge) spanning the width of each representative ECT per time point. Means and standard deviations determined as in panel C. Color images available online at www.liebertpub.com/tea
FIG. 5.
FIG. 5.
Quantification of cell orientation over time in ECT. (A, B) Confocal immunofluorescence images of representative full-width ECT sections from each group labeled with rhodamine-phalloidin at culture days 5 (A) and 20 (B). Scale bar=100 μm. (C) Example image processed with the MatFiber script used for measuring cell alignment. Mean vector length, r=0.75, in this example. (D) Mean vector length for three ECT groups from 48 h to 20 days, indicating increasing cell alignment with time in culture. Mean values and standard deviations describe data from three locations (left, middle, and right) along one representative ECT from each group per time point.
FIG. 6.
FIG. 6.
Developed stress (Pa) versus days in culture for three ECT test groups: (1) ■ baseline, (2) □ myocyte-depleted, and (3) formula image myocyte-depleted+MSC supplement. The myocyte-depleted group showed a significantly decreased developed stress compared to baseline, demonstrating that depletion of cardiomyocytes had a detrimental effect on ECT contractile properties. Supplementing myocyte-depleted ECT with a 10:1 ratio of neonatal rat cardiomyocyte:MSC resulted in restoration of contractile function with significantly enhanced developed stress at days 5 through 15 compared to the myocyte-depleted group. Sample size (n) indicated for all time points. Bars indicate standard deviation. **p≤0.01 and ***p≤0.001.
FIG. 7.
FIG. 7.
Cross-sectional area (CSA) (mm2) versus days in culture. CSA decreased from day 5 to 20, indicating tissue compaction with time in culture. Supplementation with MSCs resulted in faster compaction and a trend toward smaller CSA compared with baseline and myocyte-depleted groups. Sample size (n) as in Figure 6. *p<0.05, **p≤0.01, and ***p≤0.001.
FIG. 8.
FIG. 8.
Developed force/myocyte (μN/cell×106) versus days in culture. The myocyte-depleted+MSC group showed significant enhancement in developed force per million myocytes compared to the myocyte-depleted group at days 5, 10, and 15, and were significantly higher than baseline ECT on day 20. Sample size (n) as in Figure 6. *p<0.05 and **p≤0.01.
FIG. 9.
FIG. 9.
Pacing threshold versus days in culture for three ECT test groups. The threshold voltage gradient required for electrical pacing at 2 Hz was significantly lower in the myocyte-depleted supplemented with the MSC group compared to the myocyte-depleted group at days 5 and 10. Sample size (n) indicated for all time points. *p<0.05.
FIG. 10.
FIG. 10.
Comparison of dermal fibroblast (DF)-supplemented (formula image) versus MSC-supplemented myocyte-depleted ECT (formula image). Mean±standard deviation values for CSA, DS, and developed force on culture days 5 and 10 were normalized by corresponding values for MSC-supplemented ECT at matched time points. Developed force was significantly lower in DF-supplemented ECT versus MSC-supplemented ECT at both time points, despite greater compaction of ECT by DF at day 10, indicating cell-type specificity of functional enhancement. Sample size (n) as indicated. **p≤0.01 and ***p≤0.001.
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