Microenvironmental determinants of organized iPSC-cardiomyocyte tissues on synthetic fibrous matrices

Abstract

Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) show great potential for engineering myocardium to study cardiac disease and create regenerative therapies. However, iPSC-CMs typically possess a late embryonic stage phenotype, with cells failing to exhibit markers of mature adult tissue. This is due in part to insufficient knowledge and control of microenvironmental cues required to facilitate the organization and maturation of iPSC-CMs. Here, we employed a cell-adhesive, mechanically tunable synthetic fibrous extracellular matrix (ECM) consisting of electrospun dextran vinyl sulfone (DVS) fibers and examined how biochemical, architectural, and mechanical properties of the ECM impact iPSC-CM tissue assembly and subsequent function. Exploring a multidimensional parameter space spanning cell-adhesive ligand, seeding density, fiber alignment, and stiffness, we found that fibronectin-functionalized DVS matrices composed of highly aligned fibers with low stiffness optimally promoted the organization of functional iPSC-CM tissues. Tissues generated on these matrices demonstrated improved calcium handling and increased end-to-end localization of N-cadherin as compared to micropatterned fibronectin lines or fibronectin-coated glass. Furthermore, DVS matrices supported long-term culture (45 days) of iPSC-CMs; N-cadherin end-to-end localization and connexin43 expression both increased as a function of time in culture. In sum, these findings demonstrate the importance of recapitulating the fibrous myocardial ECM in engineering structurally organized and functional iPSC-CM tissues.

Fig. 1

Tunable DVS fibrous matrices for culturing iPSC-CMs. (a) Schematic of fabrication setup used to generate DVS matrices. A photocrosslinkable DVS polymer solution was electrospun and collected on glass coverslips affixed to a rotating hexagonal mandrel. (b and c) Mandrel rotation speed was varied to define fiber alignment. (d) To facilitate cell adhesion, fibers were functionalized with full length proteins fibronectin or collagen via a heparin sulfate conjugation scheme, or short adhesive peptides cRGD or GFOGER via Michael-type addition chemistry. (e) Fiber stiffness was tuned by altering the amount of photoinitiated crosslinking. (f) iPSCs were differentiated through temporal modulation of the Wnt signaling pathway as previously described, purified via metabolic selection for at least 4 days, and seeded on DVS fibrous substrates 30 days post initiation of differentiation. Progression of the differentiation process can be seen from brightfield images of iPSCs taken before differentiating, immunofluorescent images of iPSC-CMs just after metabolic selection, and iPSC-CMs seeded on aligned DVS matrices. All data presented as mean ± std; n ≥ 5 matrices; * p < 0.05.

Fig. 2

DVS functionalization with FN and cRGD increase cell attachment and enable formation of contractile tissues. (a) Confocal fluorescent images of iPSC-CMs cultured on aligned DVS matrices functionalized with different cell-adhesive proteins or peptides. (b) Density of resultant iPSC-CM tissues on fibers functionalized with different cell adhesion functionalization schemes. Calcium flux dynamics were analyzed to determine (c) contraction rate, (d) peak-to-peak irregularity, as quantified by the standard deviation of time interval between peaks, and (e) contraction correlation coefficient for formed tissues, as calculated by Pearson’s correlation coefficient between fluorescent profiles of subdivided regions within a field of view. All data presented as mean ± std; n ≥ 8 fields of view across 3 tissues; * p < 0.05.

Fig. 3

iPSC-CMs at intermediate seeding density form highly organized and functional tissues. (a) Confocal fluorescent images of iPSC-CMs on aligned DVS matrices functionalized with fibronectin seeded at different densities ranging from 50 to 250k cm⁻². (b) Histogram of distribution of sarcomere angle in representative tissues seeded at 50, 150, and 250k cm⁻². (c) Quantification of overall sarcomere alignment of tissues seeded at densities ranging from 50 to 250k cm⁻². (d) Beat rate, (e) peak-to-peak irregularity and (f) contraction correlation coefficient for formed tissues obtained via analysis of calcium fluxes. (g) Image slices at 0, 5, and 10 μm from substrate surface taken via confocal microscopy within a dense (250k cm⁻²) tissue. (h) Sarcomere alignment deviation of slices within specified height ranges for tissues seeded at 250k cm⁻². All data presented as mean ± std; n ≥ 8 fields of view across 3 tissues; * p < 0.05.

Fig. 4

Aligned matrices promote myofibril organization and improve tissue function. (a) Confocal fluorescent images of iPSC-CMs seeded on aligned (2100 rpm), intermediate (1100 rpm), and random (100 rpm). (b) Quantification of sarcomere alignment deviation. (c) Peak-to-peak and (d) correlation coefficient irregularity, as calculated from calcium flux data. (e) Individual calcium fluxes were analyzed to determine flux rise time, decay time, and peak full width half max. All data presented as mean ± std; n ≥ 12 fields of view across 3 tissues; * p < 0.05.

Fig. 5

iPSC-CMs on soft, DVS fibers exhibit improved calcium handling. (a) Confocal fluorescent images of iPSC-CMs seeded on aligned matrices composed of DVS fibers of differing stiffnesses by tuning photoinitiated crosslinking via photoinitiator (LAP) concentration. (b) Quantification of sarcomere alignment deviation. (c) Correlation coefficient and (d) peak-to-peak irregularity, as calculated from calcium flux data. (e) Individual calcium fluxes were analyzed to determine flux rise time, decay time, and peak full width half max. All data presented as mean ± std; n ≥ 12 fields of view across 3 tissues; * p < 0.05.

Fig. 6

Fibrous DVS matrices promote increased tissue function and organization and allow for long-term culture of confluent iPSC-CMs. (a) Confocal fluorescent images of iPSC-CMs seeded on fibronectin-coated glass, microcontact printed fibronectin lines, and aligned DVS fibers modified with adhesive fibronectin after 7 days in culture. Red arrows indicate areas of N-cadherin localization at end-end cell junctions. (b) Quantification of sarcomere angle deviation across all substrates. (c) Calcium handling quantifications of peak-to-peak irregularity, (d) contraction correlation coefficient, and (e) flux rise time, decay time, and full width half max. (f) Survival of cultures over time on various substrates. All data presented as mean ± std; n ≥ 12 fields of view across 3 tissues; * p < 0.05.

Fig. 7

Aligned fibrous matrices induce myofibril organization and improve tissue function. Confocal fluorescent images of iPSC-CMs seeded on DVS cultured for 7, 14, and 28 days with immunostaining for (a) N-cadherin and (c) connexin43. Red arrows indicate localization of N-cadherin at end-to-end cell–cell junctions. (b) Quantification of sarcomere angle deviation over time. (d) Quantification of connexin43 expression over time. (e) Calcium handling quantifications of peak-to-peak irregularity, (f) contraction correlation coefficient, and (g) flux rise time, decay time, and full width half max. All data presented as mean ± std; n ≥ 12 fields of view across 3 tissues; * p < 0.05.