Epigenetic Priming of Human Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Accelerates Cardiomyocyte Maturation.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) exhibit a fetal phenotype that limits in vitro and therapeutic applications. Strategies to promote cardiomyocyte maturation have focused interventions on differentiated hPSC-CMs, but this study tests priming of early cardiac progenitor cells (CPCs) with polyinosinic-polycytidylic acid (pIC) to accelerate cardiomyocyte maturation. CPCs were differentiated from hPSCs using a monolayer differentiation protocol with defined small molecule Wnt temporal modulation, and pIC was added during the formation of early CPCs. pIC priming did not alter the expression of cell surface markers for CPCs (>80% KDR+/PDGFRα+), expression of common cardiac transcription factors, or final purity of differentiated hPSC-CMs (∼90%). However, CPC differentiation in basal medium revealed that pIC priming resulted in hPSC-CMs with enhanced maturity manifested by increased cell size, greater contractility, faster electrical upstrokes, increased oxidative metabolism, and more mature sarcomeric structure and composition. To investigate the mechanisms of CPC priming, RNAseq revealed that cardiac progenitor-stage pIC modulated early Notch signaling and cardiomyogenic transcriptional programs. Chromatin immunoprecipitation of CPCs showed that pIC treatment increased deposition of the H3K9ac activating epigenetic mark at core promoters of cardiac myofilament genes and the Notch ligand, JAG1. Inhibition of Notch signaling blocked the effects of pIC on differentiation and cardiomyocyte maturation. Furthermore, primed CPCs showed more robust formation of hPSC-CMs grafts when transplanted to the NSGW mouse kidney capsule. Overall, epigenetic modulation of CPCs with pIC accelerates cardiomyocyte maturation enabling basic research applications and potential therapeutic uses. Stem Cells 2019;37:910-923.

Micropatterned substrates with physiological stiffness promote cell maturation and Pompe disease phenotype in human induced pluripotent stem cell-derived skeletal myocytes.

Recent advances in bioengineering have enabled cell culture systems that more closely mimic the native cellular environment. Here, we demonstrated that human induced pluripotent stem cell (iPSC)-derived myogenic progenitors formed highly-aligned myotubes and contracted when seeded on two-dimensional micropatterned platforms. The differentiated cells showed clear nuclear alignment and formed elongated myotubes dependent on the width of the micropatterned lanes. Topographical cues from micropatterning and physiological substrate stiffness improved the formation of well-aligned and multinucleated myotubes similar to myofibers. These aligned myotubes exhibited spontaneous contractions specifically along the long axis of the pattern. Notably, the micropatterned platforms developed bundle-like myotubes using patient-derived iPSCs with a background of Pompe disease (glycogen storage disease type II) and even enhanced the disease phenotype as shown through the specific pathology of abnormal lysosome accumulations. A highly-aligned formation of matured myotubes holds great potential in further understanding the process of human muscle development, as well as advancing in vitro pharmacological studies for skeletal muscle diseases.

Biocompatible, degradable thermoplastic polyurethane based on polycaprolactone-block-polytetrahydrofuran-block-polycaprolactone copolymers for soft tissue engineering.

Biodegradable synthetic polymers have been widely used as tissue engineering scaffold materials. Even though they have shown excellent biocompatibility, they have failed to resemble the low stiffness and high elasticity of soft tissues because of the presence of massive rigid ester bonds. Herein, we synthesized a new thermoplastic polyurethane elastomer (CTC-PU(BET)) using poly ester ether triblock copolymer (polycaprolactone-block-polytetrahydrofuran-block-polycaprolactone triblock copolymer, PCTC) as the soft segment, aliphatic diisocyanate (hexamethylene diisocyanate, HDI) as the hard segment, and degradable diol (bis(2-hydroxyethyl) terephthalate, BET) as the chain extender. PCTC inhibited crystallization and reduced the melting temperature of CTC-PU(BET), and BET dramatically enhanced the thermal decomposition and hydrolytic degradation rate when compared with conventional polyester-based biodegradable TPUs. The CTC-PU(BET) synthesized in this study possessed a low tensile modulus and tensile strength of 2.2 MPa and 1.3 MPa, respectively, and an elongation-at-break over 700%. Meanwhile, it maintained a 95.3% recovery rate and 90% resilience over ten cycles of loading and unloading. In addition, the TPU could be electrospun into both random and aligned fibrous scaffolds consisting of major microfibers and nanobranches. 3T3 fibroblast cell culture confirmed that these scaffolds outperformed the conventional biodegradable TPU scaffolds in terms of substrate-cellular interactions and cell proliferation. Considering the advantages of this TPU, such as ease of synthesis, low cost, low stiffness, high elasticity, controllable degradation rate, ease of processability, and excellent biocompatibility, it has great prospects to be used as a tissue engineering scaffold material for soft tissue regeneration.

Micropattern width dependent sarcomere development in human ESC-derived cardiomyocytes.

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.