Cardiac Regeneration and PLGA Nanofibers
This time, we investigated the use of PLGA nanofibers for the maturation of cardiomyocytes derived from iPS cells!
Tsumugi
She is a second-year researcher in the Nanofiber Division of MECC.
Her hobbies include reading science magazines and the latest
research papers, as well as karate. Her special skill is speaking Chinese.
Background and Objectives
Cell therapy using high-purity cardiomyocytes (CM) derived from human-induced pluripotent stem cells (iPS cells) is a promising means for cardiac regeneration. It is currently possible to efficiently produce robust cardiomyocytes (hPSC-CM) under serum-free conditions. However, hPSC-CMs obtained through 2D culture are similar to immature CMs, potentially affecting the functionality and drug responsiveness of hPSC-CMs. Therefore, in this study, we generated tissue structures with morphological characteristics similar to those of myocardium by aligning hPSC-CM-derived CMs on oriented PLGA nanofibers that mimic the natural environment.
Oriented PLGA Nanofibers
Poly(lactic-co-glycolic acid) (PLGA) is a copolymer with high biodegradability and biocompatibility, approved by the Food and Drug Administration (FDA), and widely used in the medical field. Oriented PLGA nanofibers (ANF) were used as culture scaffolds for the growth and tissue formation of iPS cell-derived cells, as they are more similar to the natural extracellular matrix compared to random PLGA nanofibers (RNF). The fiber diameter of the PLGA-oriented nanofibers used as scaffolds ranged from 500 to 2000 nm, with a thickness of 1.5 to 12 μm, mimicking collagen fiber bundles in muscle tissue. By making the nanofiber sheets thinner than conventional methods, CMs were more easily able to infiltrate them.
Experiment
Observations were made by culturing iPS cell-derived CMs on substrates made using PLGA nanofibers and polydimethylsiloxane for 14 days. CMs infiltrated and wrapped around the nanofibers, and tissues matured on the ANF. Various high-density CMs were obtained, and the thickness of the tissue could be increased by increasing the number of cells or layering multiple CM sheets. CM sheets on ANF were named 3D cardiac tissue-like constructs (CTLC) as they reproduced the in vivo arrangement indicated by highly defined three-dimensional anisotropic structures.
Comparing the electrical properties of high-density ANF (H-ANF), low-density ANF (L-ANF), RNF, and CMs cultured on flat surfaces, those grown on ANF and RNF showed larger field potential amplitudes and better cell adhesion. H-ANF demonstrated high T-wave detection channel ratios in microelectrode arrays, essential for long-term monitoring of chronic drug effects on CMs, proving the potential for CTLC long-term monitoring. To evaluate drug resistance of CTLC’s electrophysiological properties, E4031 was applied, and QT interval duration was observed. CMs cultured on nanofibers showed lower QT interval variability and less pronounced arrhythmic activity compared to CMs cultured on flat surfaces, suggesting higher electrophysiological uniformity. Drug response was also observed, indicating that CTLC faithfully reproduces the structural and functional properties of in vivo cardiac tissue, making it an excellent model for drug cardiotoxicity testing.
In an in vitro grafting experiment, two CTLCs grafted together for three days showed no clear boundary between the two samples, and the grafted tissue was twice as thick as a single CTLC layer, indicating rapid tissue integration. Additionally, CTLCs placed on two separated, independently beating host CM sheets synchronized their contractions. These results suggest that CTLCs integrate with host CMs and could facilitate rapid electrical coupling between severed regions within the heart, potentially curing reentrant arrhythmias.
Analyzing and optimizing CTLC transplantation parameters, and transplanting optimized CTLCs to the rat ventricular epicardium, showed no histological gaps. Thick clusters of human TnT-positive CMs were observed in the transplanted rat heart, with some hTnT-positive cells infiltrating the rat ventricular epicardium. Additionally, the transplanted CTLC grafts showed aligned and organized compact myocardial structures, indicating efficient CTLC transplantation and survival of iPS cell-derived CMs for 14 days post-transplantation. Furthermore, CTLCs were transplanted into the hearts of 12 rats with myocardial infarction, and cell-free nanofiber scaffolds were transplanted into 17 control rats with myocardial infarction. CTLCs were observed on the heart surface of rats at four weeks post-transplantation, whereas cell-free nanofiber scaffolds were barely visible. Small vessel density was higher in the CTLC group compared to the control group. There was no significant difference in baseline ejection fraction between the two groups at week 0, but an increase in ejection fraction was observed in the rats treated with CTLC at four weeks post-transplantation.
Conclusion
CTLCs are highly versatile and easy to handle, making them suitable for both drug evaluation and transplantation. For drug evaluation, CTLCs provide a promising model for ex vivo tissue-based drug screening, demonstrating superior responses to applied drugs and proving to be an accurate and robust model. When used for grafting, CTLCs showed excellent operability and enabled rapid coupling and suppression of reentrant arrhythmias in severed or scarred tissue blocks. Additionally, in vivo experiments using rats demonstrated the potential for myocardial infarction treatment with CTLC transplantation results in rats with myocardial infarction. Overall, CTLCs hold great potential for future use in cell-based applications such as drug testing and regenerative cardiac therapies.
Impressions
It was intriguing to see that although the nanofiber sheets were very thin, they were robust and elastic. The videos included in the paper showed the integration over time, which was fascinating to observe.
This development could expand the range of treatments and experiments, contributing further to medical advancements.
Our company has also tried making PLGA-oriented nanofiber sheets. While we didn’t achieve much elasticity, we were able to create thin, robust nanofiber sheets.
Although we didn’t achieve much elasticity, we were able to create thin and durable nanofiber sheets.
They have a glossy finish and turned out beautifully ☻
Our company produces various nanofiber samples.
If you have any materials you would like to test or any questions regarding spinning, please feel free to contact us.
■References:
Junjun Li et al.(2017)
Human Pluripotent Stem Cell-Derived Cardiac Tissue-like Constructs for Repairing the Infarcted Myocardium. Stem Cell Reports(https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(17)30414-9?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2213671117304149%3Fshowall%3Dtrue)