skip to main content

Attention:

The NSF Public Access Repository (PAR) system and access will be unavailable from 11:00 PM ET on Thursday, February 13 until 2:00 AM ET on Friday, February 14 due to maintenance. We apologize for the inconvenience.


This content will become publicly available on October 29, 2025

Title: Cellular Alignment and Matrix Stiffening Induced Changes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes
Abstract

Biological processes are inherently dynamic, necessitating biomaterial platforms capable of spatiotemporal control over cellular organization and matrix stiffness for accurate study of tissue development, wound healing, and disease. However, most in vitro platforms remain static. In this study, a dynamic biomaterial platform comprising a stiffening hydrogel is introduced and achieved through a stepwise approach of addition followed by light‐mediated crosslinking, integrated with an elastomeric substrate featuring strain‐responsive lamellar surface patterns. Employing this platform, the response of human induced pluripotent stem cell‐derived cardiomyocytes (hIPSC‐CMs) is investigated to dynamic stiffening from healthy to fibrotic tissue stiffness. The results demonstrate that culturing hIPSC‐CMs on physiologically relevant healthy stiffness significantly enhances their function, as evidenced by increased sarcomere fraction, wider sarcomere width, significantly higher connexin‐43 content, and elevated cell beating frequency compared to cells cultured on fibrotic matrix. Conversely, dynamic matrix stiffening negatively impacts hIPSC‐CM function, with earlier stiffening events exerting a more pronounced hindering effect. These findings provide valuable insights into material‐based approaches for addressing existing challenges in hIPSC‐CM maturation and have broader implications across various tissue models, including muscle, tendon, nerve, and cornea, where both cellular alignment and matrix stiffening play pivotal roles in tissue development and regeneration.

 
more » « less
Award ID(s):
2044479
PAR ID:
10561010
Author(s) / Creator(s):
; ; ; ;
Publisher / Repository:
Wiley
Date Published:
Journal Name:
Advanced Healthcare Materials
ISSN:
2192-2640
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Recent innovations in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs) have unlocked a viable path to creating in vitro cardiac models. Currently, hiPSC-derived cardiomyocytes (hiPSC-CMs) remain immature, leading many in the field to explore approaches to enhance cell and tissue maturation. Here, we systematically analyzed 300 studies using hiPSC-CM models to determine common fabrication, maturation and assessment techniques used to evaluate cardiomyocyte functionality and maturity and compiled the data into an open-access database. Based on this analysis, we present the diversity of, and current trends in, in vitro models and highlight the most common and promising practices for functional assessments. We further analyzed outputs spanning structural maturity, contractile function, electrophysiology and gene expression and note field-wide improvements over time. Finally, we discuss opportunities to collectively pursue the shared goal of hiPSC-CM model development, maturation and assessment that we believe are critical for engineering mature cardiac tissue. 
    more » « less
  2. Clinical translation of stem cell therapies for heart disease requires electrical integration of transplanted cardiomyocytes. Generation of electrically matured human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) is critical for electrical integration. Here, we found that hiPSC-derived endothelial cells (hiPSC-ECs) promoted the expression of selected maturation markers in hiPSC-CMs. Using tissue-embedded stretchable mesh nanoelectronics, we achieved a long-term stable map of human three-dimensional (3D) cardiac microtissue electrical activity. The results revealed that hiPSC-ECs accelerated the electrical maturation of hiPSC-CMs in 3D cardiac microtissues. Machine learning–based pseudotime trajectory inference of cardiomyocyte electrical signals further revealed the electrical phenotypic transition path during development. Guided by the electrical recording data, single-cell RNA sequencing identified that hiPSC-ECs promoted cardiomyocyte subpopulations with a more mature phenotype, and multiple ligand-receptor interactions were up-regulated between hiPSC-ECs and hiPSC-CMs, revealing a coordinated multifactorial mechanism of hiPSC-CM electrical maturation. Collectively, these findings show that hiPSC-ECs drive hiPSC-CM electrical maturation via multiple intercellular pathways.

     
    more » « less
  3. Rationale: Dominant heterozygous variants in filamin C ( FLNC ) cause diverse cardiomyopathies, although the underlying molecular mechanisms remain poorly understood. Objective: We aimed to define the molecular mechanisms by which FLNC variants altered human cardiomyocyte gene and protein expression, sarcomere structure, and contractile performance. Methods and Results: Using CRISPR/Cas9, we introduced FLNC variants into human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). We compared isogenic hiPSC-CMs with normal (wild-type), ablated expression ( FLNC −/− ), or haploinsufficiency ( FLNC +/− ) that causes dilated cardiomyopathy. We also studied a heterozygous in-frame deletion ( FLNC +/Δ7aa ) which did not affect FLNC expression but caused aggregate formation, similar to FLNC variants associated with hypertrophic cardiomyopathy. FLNC −/− hiPSC-CMs demonstrated profound sarcomere misassembly and reduced contractility. Although sarcomere formation and function were unaffected in FLNC +/ − and FLNC +/Δ7aa hiPSC-CMs, these heterozygous variants caused increases in lysosome content, enhancement of autophagic flux, and accumulation of FLNC-binding partners and Z-disc proteins. Conclusions: FLNC expression is required for sarcomere organization and physiological function. Variants that produce misfolded FLNC proteins cause the accumulation of FLNC and FLNC-binding partners which leads to increased lysosome expression and activation of autophagic pathways. Surprisingly, similar pathways were activated in FLNC haploinsufficient hiPSC-CMs, likely initiated by the loss of stoichiometric FLNC protein interactions and impaired turnover of proteins at the Z-disc. These results indicate that both FLNC haploinsufficient variants and variants that produce misfolded FLNC protein cause disease by similar proteotoxic mechanisms and indicate the therapeutic potential for augmenting protein degradative pathways to treat a wide range of FLNC -related cardiomyopathies. 
    more » « less
  4. null (Ed.)
    Background: The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin ( TTN ) are the leading known cause of familial dilated cardiomyopathy. The uneven distribution of these mutations within TTN motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function. Methods: To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z −/− ) and A-band (TTN-A −/− ) regions of the TTN gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements. Results: After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z −/− -CMs had sarcomeres and visibly contracted, whereas TTN-A −/− -CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z −/− -CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z −/− -CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin. Conclusions: We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to human cardiac disease. 
    more » « less
  5. The epigenetic landscape and the responses to pharmacological epigenetic regulators in each human are unique. Classes of epigenetic writers and erasers, such as histone acetyltransferases, HATs, and histone deacetylases, HDACs, control DNA acetylation/deacetylation and chromatin accessibility, thus exerting transcriptional control in a tissue- and person-specific manner. Rapid development of novel pharmacological agents in clinical testing—HDAC inhibitors (HDACi)—targets these master regulators as common means of therapeutic intervention in cancer and immune diseases. The action of these epigenetic modulators is much less explored for cardiac tissue, yet all new drugs need to be tested for cardiotoxicity. To advance our understanding of chromatin regulation in the heart, and specifically how modulation of DNA acetylation state may affect functional electrophysiological responses, human-induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM) technology can be leveraged as a scalable, high-throughput platform with ability to provide patient-specific insights. This review covers relevant background on the known roles of HATs and HDACs in the heart, the current state of HDACi development, applications, and any adverse cardiac events; it also summarizes relevant differential gene expression data for the adult human heart vs. hiPSC-CMs along with initial transcriptional and functional results from using this new experimental platform to yield insights on epigenetic control of the heart. We focus on the multitude of methodologies and workflows needed to quantify responses to HDACis in hiPSC-CMs. This overview can help highlight the power and the limitations of hiPSC-CMs as a scalable experimental model in capturing epigenetic responses relevant to the human heart. 
    more » « less