Cardiac differentiation of cardiosphere-derived cells in scaffolds mimicking morphology of the cardiac extracellular matrix

Abstract

Stem cell therapy has the potential to regenerate heart tissue after myocardial infarction (MI). The regeneration is dependent upon cardiac differentiation of the delivered stem cells. We hypothesized that timing of the stem cell delivery determines the extent of cardiac differentiation as cell differentiation is dependent on matrix properties such as biomechanics, structure and morphology, and these properties in cardiac extracellular matrix (ECM) continuously vary with time after MI. In order to elucidate the relationship between ECM properties and cardiac differentiation, we created an in vitro model based on ECM-mimicking fibers and a type of cardiac progenitor cell, cardiosphere-derived cells (CDCs). A simultaneous fiber electrospinning and cell electrospraying technique was utilized to fabricate constructs. By blending a highly soft hydrogel with a relatively stiff polyurethane and modulating fabrication parameters, tissue constructs with similar cell adhesion property but different global modulus, single fiber modulus, fiber density and fiber alignment were achieved. The CDCs remained alive within the constructs during a 1week culture period. CDC cardiac differentiation was dependent on the scaffold modulus, fiber volume fraction and fiber alignment. Two constructs with relatively low scaffold modulus, ∼50-60kPa, most significantly directed the CDC differentiation into mature cardiomyocytes as evidenced by gene expressions of cardiac troponin T (cTnT), calcium channel (CACNA1c) and cardiac myosin heavy chain (MYH6), and protein expressions of cardiac troponin I (cTnI) and connexin 43 (CX43). Of these two low-modulus constructs, the extent of differentiation was greater for lower fiber alignment and higher fiber volume fraction. These results suggest that cardiac ECM properties may have an effect on cardiac differentiation of delivered stem cells.

Keywords: Cardiac differentiation; Cardiosphere-derived cells; Matrix modulus; Stem cell therapy.

Fig. 1

Viability of CDCs electrosprayed at different voltages (10, 15 and 20 kV).

Fig. 2

Distribution of cells of the tissue construct at different depths on day 1 (a) and day 7 (b).

Fig. 3

SEM images of tissue construct cross-sections, surfaces and fiber alignment/orientation distributions calculated based on the images. (a) HG9PU1/7.5; (b) HG9PU1/4.5; (c) HG7PU3/4.5; (d) HG5PU5/4.5.

Fig. 4

Representative stress–strain curves of the tissue constructs tested at 37 °C and in an aqueous condition.

Fig. 5

AFM characterization of scaffold morphology (a) and single-fiber modulus of scaffolds (b).

Figure 6

CDC adhesion on 2-D films of hydrogel/polyurethane blends with different ratios.

Fig. 7

dsDNA content of tissue constructs after 1 and 7 days of culture in spinner flasks.

Fig. 8

Gene expression of CDCs in tissue constructs after 7 days of culture in spinner flasks. (a) CACNA1c; (b) cTnT; (c) MYH6.

Fig. 9

CX43 expression (red) of CDCs in tissue constructs after 7 days of culture in spinner flasks. Cell nuclei were stained by Hoechst (blue). (a) HG9PU1/7.5; (b) HG9PU1/4.5; (c) HG7PU3/4.5; (d) HG5PU5/4.5.

Fig. 10

cTnI expression (green) of CDCs in tissue constructs after 7 days of culture in spinner flasks. Cell nuclei were stained by Hoechst (blue). (a) HG9PU1/7.5; (b) HG9PU1/4.5; (c) HG7PU3/4.5; (d) HG5PU5/4.5.

Fig. 11

Simulated relationship of gene/protein expression (GP), fiber volume fraction (V_f), and small strain scaffold modulus (M₀).