Maturation of three-dimensional, hiPSC-derived cardiomyocyte spheroids utilizing cyclic, uniaxial stretch and electrical stimulation

Abstract

Functional myocardium derived from human induced pluripotent stem cells (hiPSCs) can be impactful for cardiac disease modeling, drug testing, and the repair of injured myocardium. However, when hiPSCs are differentiated into cardiomyocytes, they do not possess characteristics of mature myocytes which limits their application in these endeavors. We hypothesized that mechanical and electrical stimuli would enhance the maturation of hiPSC-derived cardiomyocyte (hiPSC-CM) spheroids on both a structural and functional level, potentially leading to a better model for drug testing as well as cell therapy. Spheroids were generated with hiPSC-CM. For inducing mechanical stimulation, they were placed in a custom-made device with PDMS channels and exposed to cyclic, uniaxial stretch. Spheroids were electrically stimulated in the C-Pace EP from IONOptix for 7 days. Following the stimulations, the spheroids were then analyzed for cardiomyocyte maturation. Both stimulated groups of spheroids possessed enhanced transcript and protein expressions for key maturation markers, such as cTnI, MLC2v, and MLC2a, along with improved ultrastructure of the hiPSC-CMs in both groups with enhanced Z-band/Z-body formation, fibril alignment, and fiber number. Optical mapping showed that spheroids exposed to electrical stimulation were able to capture signals at increasing rates of pacing up to 4 Hz, which failed in unstimulated spheroids. Our results clearly indicate that a significantly improved myocyte maturation can be achieved by culturing iPSC-CMs as spheroids and exposing them to cyclic, uniaxial stretch and electrical stimulation.

3D Co-culture of hiPSC-Derived Cardiomyocytes With Cardiac Fibroblasts Improves Tissue-Like Features of Cardiac Spheroids

“…We have investigated spontaneous APs in 2D-cultured hiPSC-CMs, and in cardiac spheroids with and without CF. It has been hypothesized that 3D culture induces a more mature electric phenotype, although in those studies additional interventions were performed such as contractility training or different media formulations (Ronaldson-Bouchard et al, 2018;LaBarge et al, 2019;Valls-Margarit et al, 2019). Resting membrane potential in immature cardiomyocytes has been reported around −60 and −90 mV in adult cells (Barbuti et al, 2016;Zhao et al, 2018), which is in agreement with our results in monolayer-cultured hiPSC-CMs.…”

“…Similar ultrastructural features as we have found here in spontaneously contracting spheroids have also been observed in electrically paced cardiac spheroids made of hiPSC-CMs (LaBarge et al, 2019). Although the model used here shows spontaneous activity over almost the entire culture time, this does not seem to be sufficient for a substantial maturation of the cells.…”

A Brief Review of Current Maturation Methods for Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes

“…Moderate afterloads are beneficial on cardiomyocyte maturation, while higher afterloads may be detrimental and cause pathological changes (Leonard et al, 2018). The combinations of both electrical and mechanical stimuli -either cyclic stretch or static stress -were also explored (Ruan et al, 2016;LaBarge et al, 2019). In such conditions, hiPSC-CMs displayed a more matured signature than a single stimulus.…”

Maturation strategies and limitations of induced pluripotent stem cell-derived cardiomyocytes

“…The extra electrical stimulation can increase the cell's contractility, and the cell's ability to process Ca 2+ is more mature. In addition, electrical stimulation can also increase the expression of gap junction proteins (Cx43), N-cadherin (N-Cad), improve the integration between them and host cells, and significantly improve mitochondrial alignment [73,76].…”