skip to main content

Attention:

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


Title: Engineering anisotropic human stem cell-derived three-dimensional cardiac tissue on-a-chip
Despite significant efforts in the study of cardiovascular diseases (CVDs), they persist as the leading cause of mortality worldwide. Considerable research into human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) has highlighted their immense potential in the development of in vitro human cardiac tissues for broad mechanistic, therapeutic, and patient-specific disease modeling studies in the pursuit of CVD research. However, the relatively immature state of hPSC-CMs remains an obstacle in enhancing clinical relevance ofengineered cardiac tissue models. In this study, we describe development of a microfluidic platform for 3D modeling of cardiac tissues, derived from both rat cells and hPSC-CMs, to better recapitulate the native myocardium through co-culture with interstitial cells (specifically cardiac fibroblasts), biomimetic collagen hydrogel encapsulation, and induction of highly anisotropic tissue architecture. The presented platform is precisely engineered through incorporation of surface topography in the form of staggered microposts to enable long-term culture and maturation of cardiac cells, resulting in formation of physiologically relevant cardiac tissues with anisotropy that mimics native myocardium. After two weeks of culture, hPSC-derived cardiac tissues exhibited well-defined sarcomeric striations, highly synchronous contractions, and upregulation of several maturation genes, including HCN1, KCNQ1, CAV1.2, CAV3.1, PLN, and RYR2. These findings demonstrate the ability of the proposed engineered platform to mature animal- as well as human stem cell-derived cardiac tissues over an extended period of culture, providing a novel microfluidic chip with the capability for cardiac disease modeling and therapeutic testing.  more » « less
Award ID(s):
1653193
PAR ID:
10212395
Author(s) / Creator(s):
; ; ; ;
Date Published:
Journal Name:
Biomaterials
Volume:
256
Issue:
120195
ISSN:
0142-9612
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) have emerged as an exciting new tool for cardiac research and can serve as a preclinical platform for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC‐CMs resemble fetal human cardiomyocytes in terms of structure and function. Herein we provide a novel in vitro platform that includes patterned extracellular matrix with physiological substrate stiffness and is amenable to both mechanical and electrical analysis. Micropatterned lanes promote the cellular and myofibril alignment of hPSC‐CMs while the addition of micropatterned bridges enable formation of a functional cardiac syncytium that beats synchronously over a large two‐dimensional area. We investigated the electrophysiological properties of the patterned cardiac constructs and showed they have anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the primary direction of the pattern as compared to the transverse direction. Lastly, we interrogated the mechanical function of the pattern constructs and demonstrated the utility of this platform in recording the strength of cardiomyocyte contractions. This biomimetic platform with electrical and mechanical readout capabilities will enable the study of cardiac disease and the influence of pharmaceuticals and toxins on cardiomyocyte function. The platform also holds potential for high throughput evaluation of drug safety and efficacy, thus furthering our understanding of cardiovascular disease and increasing the translational use of hPSC‐CMs.

     
    more » « less
  2. Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are immature in their structure and function, limiting their potential in disease modeling, drug screening, and cardiac cellular therapies. Prior studies have demonstrated that coculture of hPSC‐derived CMs with other cardiac cell types, including endothelial cells (ECs), can accelerate CM maturation. To address whether the CM differentiation stage at which ECs are introduced affects CM maturation, the authors coculture hPSC‐derived ECs with hPSC‐derived cardiac progenitor cells (CPCs) and CMs and analyze the molecular and functional attributes of maturation. ECs have a more significant effect on acceleration of maturation when cocultured with CPCs than with CMs. EC coculture with CPCs increases CM size, expression of sarcomere, and ion channel genes and proteins, the presence of intracellular membranous extensions, and chronotropic response compared to monoculture. Maturation is accelerated with an increasing EC:CPC ratio. This study demonstrates that EC incorporation at the CPC stage of CM differentiation expedites CM maturation, leading to cells that may be better suited for in vitro and in vivo applications of hPSC‐derived CMs.

     
    more » « less
  3. Abstract

    The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR-90 line) were differentiated to hPSC-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin+ flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 106 hPSC-CMs were mixed with 0.4 × 106 human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable with rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, β-adrenergic receptors, and t-tubule protein caveolin-3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering.

     
    more » « less
  4. Abstract

    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) allow investigations in a human cardiac model system, but disorganized mechanics and immaturity of hPSC-CMs on standard two-dimensional surfaces have been hurdles. Here, we developed a platform of micron-scale cardiac muscle bundles to control biomechanics in arrays of thousands of purified, independently contracting cardiac muscle strips on two-dimensional elastomer substrates with far greater throughput than single cell methods. By defining geometry and workload in this reductionist platform, we show that myofibrillar alignment and auxotonic contractions at physiologic workload drive maturation of contractile function, calcium handling, and electrophysiology. Using transcriptomics, reporter hPSC-CMs, and quantitative immunofluorescence, these cardiac muscle bundles can be used to parse orthogonal cues in early development, including contractile force, calcium load, and metabolic signals. Additionally, the resultant organized biomechanics facilitates automated extraction of contractile kinetics from brightfield microscopy imaging, increasing the accessibility, reproducibility, and throughput of pharmacologic testing and cardiomyopathy disease modeling.

     
    more » « less
  5. Cardiovascular disease is the leading cause of death worldwide and bears an immense economic burden. Late-stage heart failure often requires total heart transplantation; however, due to donor shortages and lifelong immunosuppression, alternative cardiac regenerative therapies are in high demand. Human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells, have emerged as a viable source of human cardiomyocytes for transplantation. Recent developments in several mammalian models of cardiac injury have provided strong evidence of the therapeutic potential of hPSC-derived cardiomyocytes (hPSC-CM), showing their ability to electromechanically integrate with host cardiac tissue and promote functional recovery. In this review, we will discuss recent developments in hPSC-CM differentiation and transplantation strategies for delivery to the heart. We will highlight the mechanisms through which hPSC-CMs contribute to heart repair, review major challenges in successful transplantation of hPSC-CMs, and present solutions that are being explored to address these limitations. We end with a discussion of the clinical use of hPSC-CMs, including hurdles to clinical translation, current clinical trials, and future perspectives on hPSC-CM transplantation. 
    more » « less