Conductive electrospun polymer improves stem cell-derived cardiomyocyte function and maturation

Abstract

Despite numerous efforts to generate mature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), cells often remain immature, electrically isolated, and may not reflect adult biology. Conductive polymers are attractive candidates to facilitate electrical communication between hPSC-CMs, especially at sub-confluent cell densities or diseased cells lacking cell-cell junctions. Here we electrospun conductive polymers to create a conductive fiber mesh and assess if electrical signal propagation is improved in hPSC-CMs seeded on the mesh network. Matrix characterization indicated fiber structure remained stable over weeks in buffer, scaffold stiffness remained near in vivo cardiac stiffness, and electrical conductivity scaled with conductive polymer concentration. Cells remained adherent and viable on the scaffolds for at least 5 days. Transcriptomic profiling of hPSC-CMs cultured on conductive substrates for 3 days showed upregulation of cardiac and muscle-related genes versus non-conductive fibers. Structural proteins were more organized and calcium handling was improved on conductive substrates, even at sub-confluent cell densities; prolonged culture on conductive scaffolds improved membrane depolarization compared to non-conductive substrates. Taken together, these data suggest that blended, conductive scaffolds are stable, supportive of electrical coupling in hPSC-CMs, and promote maturation, which may improve our ability to model cardiac diseases and develop targeted therapies.

Keywords: Calcium handling; Desmoplakin; FluoVolt; Sarcomere organization; poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS); poly(vinyl) alcohol (PVA).

Fig. 1.

Physical characterization of electrospun scaffolds. (a) SEM images of 8 % PVA electrospun scaffolds incubated in DPBS 4 weeks post-crosslinking with glutaraldehyde vapor for 15 min, (b) 30 min, and (c) 30 min with 9.75 % PEDOT:PSS/PVA. Scale bar = 2 μm. (d) After 4 weeks of incubation, 8 % PVA crosslinked for 15 min had a median fiber diameter of 828.04 nm (n = 3 scaffolds for week 0, 1, 4; n = 4 scaffolds for week 2; 3 average measurements per scaffold), while 8 % PVA crosslinked for 30 min had a median fiber diameter of 302.7 nm and 560.1 nm with PEDOT:PSS (n = 4 scaffolds per timepoint, 3 average measurements per scaffold). (e) Bulk stiffness (kPa) of PVA-only (8 % and 11 %) and 8 % PVA with PEDOT:PSS (3.25 %, 6.5 %, and 9.75 % mass ratio) electrospun scaffolds crosslinked for 30 min measured by AFM. Median stiffness values of 8 % PVA, 11 % PVA, 3.25 %, 6.5 %, and 9.75 % were 9.30, 26.68, 18.36, 27.91, and 44.76 kPa, respectively (n = 4 scaffolds per condition; 1 point represents 1 force map). (f) Conductivity measurements of 8 % PVA electrospun scaffolds containing 0–9.75 % PEDOT:PSS measured by TLM. Median conductivity values of 3.25 %, 6.5 %, and 9.75 % were 1.09 × 10⁻⁷, 1.57 × 10⁻⁵, and 7.00 × 10⁻³ S/cm², respectively. Each point indicates one scaffold (n.c. indicates not conductive, n = 2). Statistical significance was determined via a mixed-effects analysis (d), a one-way ANOVA with Tukey’s multiple comparison test (e), or just a one-way ANOVA (f).

Fig. 2.

Cells are viable and proliferative independent of PEDOT:PSS concentration. LIVE/DEAD staining 1 and 5 days after NIH/3T3 cell seeding on (a) glass, (b) 8 % and 11 % PVA, and (c) 8 % PVA scaffolds containing 3.25 % and 9.75 % PEDOT:PSS. Green indicates live cells and red indicates dead cells. Scale bars (a–c) = 100 μm. (d) Cell viability was calculated as the number of live cells over the total (live + dead) cells and represented as mean ± std dev. At day 5, cell viability is above 90 % for glass and electrospun scaffolds containing PEDOT:PSS. For D1, n = 2, 3, 2, 3, and 3 for glass, 8 % and 11 % PVA, and 8 % PVA scaffolds containing 3.25 % and 9.75 % PEDOT:PSS, respectively. For D5, n = 2, 3, 3, 4, and 4 for glass, 8 % and 11 % PVA, and 8 % PVA scaffolds containing 3.25 % and 9.75 % PEDOT:PSS, respectively. (e) Cell proliferation (mean ± std) as measured by resazurin fluorescence intensity illustrates higher cell viability with increasing PEDOT:PSS concentration as compared to PVA-only scaffolds. For D1, n = 1, 4, 2, 4, and 4 for glass, 8 % and 11 % PVA, and 8 % PVA scaffolds containing 3.25 % and 9.75 % PEDOT: PSS, respectively. For D5, n = 1, 4, 2, 4, and 4 for glass, 8 % and 11 % PVA, and 8 % PVA scaffolds containing 3.25 % and 9.75 % PEDOT:PSS, respectively. (f) The fraction of Ki67 positive cells is indicated in the plot at day 5 post-plating on the indicated scaffolds. Statistical significance was determined via a mixed-effects analysis for (d), a two-way ANOVA for (e), or a one-way ANOVA for (f), both with Tukey’s multiple comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3.

RNA sequencing identifies upregulation of cardiac-related genes on conductive substrates. (a) PCA (post-batch correction) for hPSC-CMs seeded on 8 % PVA or 8 % PVA+9.75 % PEDOT:PSS scaffolds with total contribution from each axis noted (left) and Euclidian distance matrix (right) indicating the similarity between groups, where 30 indicates the largest dissimilarity between samples. (b) Volcano plot (left) indicating differences in DEGs between 8 % PVA and conductive scaffolds and heatmap (right) illustrating transcriptomic clustering of the top 287 significant genes. (c) Top four GO terms for each experimental condition and their statistical significance. (d) Normalized expression of genes related to hPSC-CM contractility, sarcomere organization, and electrophysiology (p-adj. < 0.05). A FC cut-off >0.25 and p-adj. cut-off <0.05 for (b) and (c) was used.

Fig. 4.

Structural characterization of hPSC-CMs. (a) IF images of desmoplakin (DP) localization in hPSC-CMs seeded on glass, 8 % PVA, and PEDOT:PSS/PVA (3.25 % and 9.75 %) electrospun scaffolds at the indicated densities. Scale bar = 20 μm. (b) Mean DP pixel intensity is plotted for each condition. Data is reported as the mean pixel intensity of DP across four biological replicates with 5–11 images per sample. (c) IF images of hPSC-CMs seeded on glass, 8 % PVA, and PEDOT:PSS/PVA (3.25 % and 9.75 %) electrospun scaffolds at the indicated densities. Filled arrowheads indicate organized sarcomeres and open arrowheads indicate unorganized sarcomeres. Scale bar = 10 μm. (d) Sarcomere organization index is plotted for the indicated scaffold compositions on a scale of 0 (least) to 1 (most) organized. Each point represents one section of an image across three biological replicates. Statistical significance was determined via a one-way ANOVA with Tukey’s multiple comparisons test (b) and Kruskal-Wallis test with Dunn’s multiple comparisons (d) test to account for unequal variance between experimental groups.

Fig. 5.

hPSC-CM calcium handling on conductive PEDOT:PSS/PVA electrospun scaffolds. (a) Graphical illustration of relevant calcium handling parameters. (b) Kymograph of hPSC-CMs in monolayer on 8 % PVA or 9.75 % PEDOT:PSS/PVA. Black chevron indicates calcium peak. Scale = 3 s dF/F_o (c) increases and time to peak (d) decreases with increasing PEDOT:PSS concentration in both low and high hPSC-CM seeding densities. Cycle length (e) and width (f) decrease with increasing PEDOT:PSS concentration at high seeding densities. Each point represents one cell across n = 3 biological replicates. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test.

Fig. 6.

hPSC-CM action potentials on conductive PEDOT:PSS/PVA electrospun scaffolds. (a) Representative images of hPSC-CMs seeded in monolayer and stained with voltage sensitive dye FluoVolt after 7 days of culture on 8 % PVA or 9.75 % PEDOT:PSS/PVA. Scale = 100 μm (b) Kymograph of hPSC-CMs ROIs on 8 % PVA or 9.75 % PEDOT:PSS/PVA. Black chevron indicates action potential peak. Scale = 3 s. (c) dF/F_o was measured on conductive scaffolds and plotted relative to glass and 8 % PVA. (d) Cycle length for rhythmically contracting cells was measured from kymographs and plotted. Since not all samples were rhythmically contracting, total percentage of depolarizing ROIs was also plotted (green) with the most number of depolarizing ROIs on conductive scaffolds (glass = 41.2 %; 8 % PVA = 42.3 %; 3.25 % = 94.4 %; 9.75 % = 100 %). Each point represents one ROI across n = 3 biological replicates. Statistical significance was determined by Kruskal-Wallis test with Dunn’s multiple comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)