Engineering Alignment Has Mixed Effects on Human Induced Pluripotent Stem Cell Differentiated Cardiomyocyte Maturation

Abstract

The potential of human induced pluripotent stem cell differentiated cardiomyocytes (hiPSC-CMs) is greatly limited by their functional immaturity. Strong relationships exist between CM structure and function, leading many in the field to seek ways to mature hiPSC-CMs by culturing on biomimetic substrates, specifically those that promote alignment. However, these in vitro models have so far failed to replicate the alignment that occurs during cardiac differentiation. We show that engineered alignment, incorporated before and during cardiac differentiation, affects hiPSC-CM electrochemical coupling and mitochondrial morphology. We successfully engineer alignment in differentiating hiPSCs as early as Day 4. We uniquely apply optical redox imaging to monitor the metabolic changes occurring during cardiac differentiation. We couple this modality with cardiac-specific markers, which allows us to assess cardiac metabolism in heterogeneous cell populations. The engineered alignment drives hiPSC-CM differentiation towards the ventricular compact CM subtype and improves electrochemical coupling in the short term. Moreover, we observe glycolysis to oxidative phosphorylation switch throughout differentiation and CM development. On the subcellular scale, we note changes in mitochondrial morphology in the long term. Our results demonstrate that cellular alignment accelerates hiPSC-CM maturity and emphasizes the interrelation of structure and function in cardiac development. We anticipate that combining engineered alignment with additional maturation strategies will result in improved development of mature CMs from hiPSC and strongly improve cardiac tissue engineering.

Integrated Manufacturing of Suspended and Aligned Nanofibrous Scaffold for Structural Maturation and Synchronous Contraction of HiPSC-Derived Cardiomyocytes

“…Furthermore, as it has been reported in the literature that myosin (encoded by the MYH gene) is a hexameric enzyme that hydrolyzes ATP [ 52 ], spontaneous cell contraction requires a large amount of myosin to provide energy, leading to the upregulation of the MYH7 gene, which correlates with the result that anisotropic structural scaffolds will affect hiPSC-CM electrochemical coupling and mitochondrial morphology [ 53 ]. On the other hand, cells contracted along the direction of aligned fibers, and myosin alignment, were highly ordered in the direction of myosin, which allowed the contraction–diastole process to generate forces in the same primary direction, and this enhanced the overall contractile force of myocardial tissue [ 54 ].…”

Dynamic Electrical Stimulation Promotes hiPSC-CM Differentiation and Functionality

“…Quantitative PCR was performed using PrimeTime qPCR Primers (Integrated DNA Technologies) and PowerUp SYBR Green (Thermo Fisher) on a StepOnePlus Real-Time PCR System (Applied Biosystems) based on previously described methods. 27 TATA-binding protein (TBP) was used as the endogenous control, and mRNA from CMs not subjected to stimulation was used as the reference sample. The full list of primers used is available in supplementary table S1.…”

“…Images were processed using a custom MALTAB code to assess degree of synchronicity, calcium cycle duration, and decay constant, as previously discussed. 27 Images were also processed with the Optical Flow Analysis Toolbox in MATLAB for Mesoscale brain activity (OFAMM) to derive calcium flow directionality and velocity. 28 Briefly, image sequences were pre-processed using median spatial filters (3X3 pixels) and low pass (<1Hz) temporal filters as recommended.…”