Environmental Cues Facilitate Maturation and Patterning of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Background/aims: Advances in induced pluripotent stem cell (iPSC) technology allow for reprogramming of adult somatic cells into stem cells from which patient- and disease-specific cardiomyocytes (CMs) can be derived. Yet, the potential of iPSC technology to revolutionize cardiovascular research is limited, in part, by the embryonic nature of these cells. Here, we test the hypothesis that decellularized porcine left ventricular extracellular cardiac matrix (ECM) provides environmental cues that promote transcriptional maturation and patterning of iPSC-CMs in culture.

Methods: Cardiac progenitor cells were plated on ECM or standard tissue plates (2D monolayer) for 30 days, after which CM orientation and single cell transcriptomics were evaluated using confocal imaging and singe cell RNA-sequencing, respectively.

Results: Cardiac progenitors differentiated on left ventricular ECM formed longitudinal fibers that differed quantitatively from progenitors differentiated in standard 2D conditions. Unsupervised clustering of single cell transcriptomics identified a CM cluster expressing a higher level of genes related to CM maturation. CMs differentiated on ECM were overrepresented in this cluster, indicating a bias toward CM maturation, compared to cells differentiated in standard 2D monolayer conditions.

Conclusion: Our data suggest that environmental cues related to the left ventricular ECM may promote differentiation to a more mature CM state compared to cells differentiated on a standard 2D monolayer, while facilitating organization into longitudinal micro-fibers. Our study highlights the utility of ECM as a differentiation substrate to promote CM maturation and fiber orientation in vitro .

Keywords: Human induced pluripotent stem cells ; Cardiomyocytes ; Extracellular matrix ; Maturation.

Fig. 1.

Experimental protocols. (A) Schematic of the iPSC reprogramming and CM differentiation protocol. (B) Plating strategy and data acquisition methods.

Fig. 2.

Macro- and micro-structural features of the left ventricular extracellular matrix (ECM). (A) Light microscopy image showing ECM macrostructure after decellularization. The resulting membrane is translucent and comprised of large fibers. (B, C.) ECM microstructure by SEM reveals the collagen fibers and tunnels that offer support to CMs differentiation and development. Scale bars 2 mm(A), 75 μm (B) and 10 μm (C).

Fig. 3.

Cardiac progenitors plated on left ventricular ECM formed longitudinal strands of CMs and fibroblasts. Top views of 3D reconstructions of 2D monolayers (A) or ECM (B) samples. iPSC-CMs (cTNT, red) differentiated on standard tissue culture plates formed clusters of cells that interconnected via strands, with fibroblasts (Vimentin, green) lacking orientation. In contrast, in ECM samples CMs formed longitudinal fibers with adjacent stands of fibroblasts. Lower panels show a 45° perspective view of the same samples highlighting the thickness and fibers in the ECM sample. Scale bar = 100μm.

Fig. 4.

Quantification of linear strands using measurements of orientation angles. The orientation of CMs for each image of the confocal z-stack was measured using the cTNT signal. Orientation angle of 0 degrees represents a straight line. Deviation from a straight line is represented by orientation angles in the positive and negative directions. Orientation angles of 2D monolayer CMs (A) were widely scattered, indicating the absence of a specific linearity across the image. N = 9 monolayer preparations and 210 z-stack slices. By contrast, the orientation angles of CMs differentiated on the ECM (B) were highly centered on 0 degrees, indicating a linear orientation of fibers across the ECM (N = 7 ECM preparations and 240 z-stack slices).

Fig. 5.

Single cell transcriptome of 2D and ECM samples. t-distributed stochastic neighbor embedding (tSNE) plots of combined 2D and ECM cultured cells. Each dot represents a single cell labelled according to culture condition (A) or cell type (B), with COL1A1 and VIM labelling fibroblasts and RYR2 and TNNT2 labelling CMs.

Fig. 6.

CMs differentiated on left ventricular ECM were enriched for genes related to cardiac development and contraction. Single cell RNA-Seq analyses comparing the CM transcriptome (as defined by RYR2 expression) for 2D monolayer and ECM differentiation protocols. (A) t-SNE plot of single CM cell transcriptomes for 2D monolayer (red) and ECM differentiation protocol (blue). (B) The same t-SNE plot as in (A), using unsupervised clustering to identify 4 unique clusters (0–3).