Micropattern width dependent sarcomere development in human ESC-derived cardiomyocytes

Abstract

In this study, human embryonic stem cell-derived cardiomyocytes were seeded onto controlled two-dimensional micropatterned features, and an improvement in sarcomere formation and cell alignment was observed in specific feature geometries. High-resolution photolithography techniques and microcontact printing were utilized to produce features of various rectangular geometries, with areas ranging from 2500 μm(2) to 160,000 μm(2). The microcontact printing method was used to pattern non-adherent poly(ethylene glycol) regions on gold coated glass slides. Matrigel and fibronectin extracellular matrix (ECM) proteins were layered onto the gold-coated glass slides, providing a controlled geometry for cell adhesion. We used small molecule-based differentiation and an antibiotic purification step to produce a pure population of immature cardiomyocytes from H9 human embryonic stem cells (hESCs). We then seeded this pure population of human cardiomyocytes onto the micropatterned features of various sizes and observed how the cardiomyocytes remodeled their myofilament structure in response to the feature geometries. Immunofluorescence was used to measure α-actinin expression, and phalloidin stains were used to detect actin presence in the patterned cells. Analysis of nuclear alignment was also used to determine how cell direction was influenced by the features. The seeded cells showed clear alignment with the features, dependent on the width rather than the overall aspect ratio of the features. It was determined that features with widths between 30 μm and 80 μm promoted highly aligned cardiomyocytes with a dramatic increase in sarcomere alignment relative to the long axis of the pattern. This creation of highly-aligned cell aggregates with robust sarcomere structures holds great potential in advancing cell-based pharmacological studies, and will help researchers to understand the means by which ECM geometries can affect myofilament structure and maturation in hESC-derived cardiomyocytes.

Keywords: Cardiac tissue engineering; Cardiomyocyte; Cell morphology; Micropatterning; Stem cell; Surface modification.

Fig. 1

Purification of hESC-derived cardiomyocyte population. The zeocin resistance of cTnT-expressing hESC-CMs was used to purify out a population of ~98% cardiomyocytes. Daily flow cytometry (A–B) indicated that this was achieved after three days of zeocin purification (n = 3). Epifluorescent imaging after purification reinforced this by showing that all visible cells were positive for cTnT-GFP protein (C).

Fig. 2

Micropattern designs. Extracellular matrix proteins were micropatterned in rectangles of varying sizes and aspect ratios. Two types of patterns were formed. (A) Features were grouped by surface area; while the features shown have varying aspect ratios, they would all have the same surface area within the same grouping. The dimensions of features within each grouping are shown in table 1. (B) Once it was determined that lane width was more important in eliciting a biological response than aspect ratio, long lanes of varying widths were formed. These lanes provided a more efficient use of space and were long enough to enable observation of calcium propagation through the hESC-CM aggregates. Dimensions shown indicate the thickness of each lane. Various spacings between neighboring lanes were attempted.

Fig. 3

Calcium transient analysis of hESC-CMs seeded onto lanes of varying widths. Live fluorescent imaging was conducted, and intensities were measured for ROIs at opposite ends of each lane (A). By comparing normalized calcium transient intensity over time (B) between the two ROIs, and using the distance between the ROIs, the calcium propagation rate was determined for each micropattern width. ROI=region of interest, FN=fibronectin.

Fig. 4

Representative brightfield images of micropatterned hESC-CMs. Cells seeded onto the micropatterned matrigel-fibronectin features matched the printed geometries very closely. (A) The first pattern design produced hESC-CM aggregates of varying aspect ratios (cells shown at day 7). All aggregates within a single grouping, as shown above, presented the same surface area. (B) The second design produced long lanes of varying widths (cells shown at day 3). As shown, the cells bound tightly to these geometric constraints and aligned clearly along the direction of the patterns. (C–D) The alignment of cells was clearly dependent on feature width, with the outermost cells aligning significantly more than cells located toward the center of each aggregate (cells shown at days 2 and 3, respectively)

Fig. 5

α-actinin, actin, and DAPI stains of cardiomyocytes after 5 days of culture on features of varying widths. Both myofilament markers show clearly improved cellular alignment, sarcomere organization, and sarcomere alignment in features under ~100 μm in width. As anticipated, cells seeded onto square features (200 μm in this set) show no favor towards any specific direction. A slight difference of 0 – 5 μm was observed between the designed pattern width and the actual width of the patterned cells. This is likely due to a small amount of swelling in the PDMS stamps during the curing step, as well as some mild mismatch between the feature edges and the cells’ exact boundaries.

Fig. 6

Connexin 43 expression of pure hESC-CMs seeded onto micropatterned matrigel/FN features of varying widths. Connexin 43 immunostaining with DAPI (A) shows a highly punctate and variable expression of gap junction plaques within the aggregates. Gap junction plaques were present along both lateral and axial edges of cardiomyocytes. A diagram (B) is included to indicate cell boundaries, as determined by phalloidin co-stain.

Fig. 7

Percentage of aligned nuclei of cells on varying features. It is clear that nuclei showed much stronger alignment on features with widths of 30 μm – 80 μm. The aspect ratios of the features as a whole, however, did little to affect the alignment of the hESC-CMs.

Fig. 8

Calcium propagation speeds of hESC-CM aggregates on matrigel/fibronectin lanes with varying widths. Rhod-2 AM ester indicator dye was used to determine the rate at which calcium waves propagated through the cardiomyocyte aggregates. A compilation of all assessed lane widths indicates that there was no clear trend between lane width and calcium transient propagation rate. Calcium propagation rates typically ranged between 0.5 cm/sec and 2 cm/sec.