Tri-culture of spatially organizing human skeletal muscle cells, endothelial cells, and fibroblasts enhances contractile force and vascular perfusion of skeletal muscle tissues.

Constructing engineered human skeletal muscle tissues that resemble the function and microstructure of human skeletal muscles is key to utilizing them in a variety of applications such as drug development, disease modeling, regenerative medicine, and engineering biological machines. However, current in vitro skeletal muscle tissues are far inferior to native muscles in terms of contractile function and lack essential cues for muscle functions, particularly heterotypic cell-cell interactions between myoblasts, endothelial cells, and fibroblasts. Here, we develop an engineered muscle tissue with a coaxial three-layered tubular structure composed of an inner endothelial cell layer, an endomysium-like layer with fibroblasts in the middle, and an outer skeletal muscle cell layer, similar to the architecture of native skeletal muscles. Engineered skeletal muscle tissues with three spatially organized cell types produced thicker myotubes and lowered Young's modulus through extracellular matrix remodeling, resulting in 43% stronger contractile force. Furthermore, we demonstrated that fibroblasts localized in the endomysium layer induced angiogenic sprouting of endothelial cells into the muscle layer more effectively than fibroblasts homogeneously distributed in the muscle layer. This layered tri-culture system enables a structured spatial configuration of the three main cell types of skeletal muscle and promotes desired paracrine signaling, resulting in improved angiogenesis and increased contractile force. This research offers new insights to efficiently obtain new human skeletal muscle models, transplantable tissues, and actuators for biological machines.

Progress in multicellular human cardiac organoids for clinical applications.

Advances in self-organizing cardiac organoids to recapitulate human cardiogenesis have provided a powerful tool for unveiling human cardiac development, studying cardiovascular diseases, testing drugs, and transplantation. Here, we highlight the recent remarkable progress on multicellular cardiac organoids and review the current status for their practical applications. We then introduce key readouts and tools for assessing cardiac organoids for clinical applications, address major challenges, and provide suggestions for each assessment method. Lastly, we discuss the current limitations of cardiac organoids as miniature models of the human heart and suggest a direction for moving forward toward building the mini-heart from cardiac organoids.

Multiscale engineered human skeletal muscles with perfusable vasculature and microvascular network recapitulating the fluid compartments.

Creating a vasculature in engineered human skeletal muscle tissues (ehSMTs) enables us to create thick tissues, increase cell survival in implantation, provide models of blood-organ barriers for drug testing, and enhance muscle differentiation through paracrine signaling. Here, contractile ehSMTs with a central perfusable vascular channel and microvascular networks growing from this central vasculature into the surrounding skeletal muscle tissue were newly demonstrated. Because coculturing muscle cells and endothelial cells requires incompatible media, we recapitulated thein vivoextracellular fluid compartments between blood plasma and interstitial fluid by creating anin vitroperfusable vasculature running through skeletal muscle tissue with a physiologic cell density. By using this model, we constructed large vascularized ehSMTs and showed the potential to be utilized for drug testing platforms. Also, we found that coculturing with two separate media from an early stage of muscle differentiation led to increased contractile force, thicker myotubes, and improved muscle differentiation.

Endothelial-Myocardial Angiocrine Signaling in Heart Development.

Vascular endothelial cells are a multifunctional cell type with organotypic specificity in their function and structure. In this review, we discuss various subpopulations of endothelial cells in the mammalian heart, which spatiotemporally regulate critical cellular and molecular processes of heart development via unique sets of angiocrine signaling pathways. In particular, elucidation of intercellular communication among the functional cell types in the developing heart has recently been accelerated by the use of single-cell sequencing. Specifically, we overview the heterogeneic nature of cardiac endothelial cells and their contribution to heart tube and chamber formation, myocardial trabeculation and compaction, and endocardial cushion and valve formation via angiocrine pathways.

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Extracellular matrix remodelling induced by alternating electrical and mechanical stimulations increases the contraction of engineered skeletal muscle tissues.

Engineered skeletal muscles are inferior to natural muscles in terms of contractile force, hampering their potential use in practical applications. One major limitation is that the extracellular matrix (ECM) not only impedes the contraction but also ineffectively transmits the forces generated by myotubes to the load. In the present study, ECM remodelling improves contractile force in a short time, and a coordinated, combined electrical and mechanical stimulation induces the desired ECM remodelling. Notably, the application of single and combined stimulations to the engineered muscles remodels the structure of their ECM networks, which determines the mechanical properties of the ECM. Myotubes in the tissues are connected in parallel and in series to the ECM. The stiffness of the parallel ECM must be low not to impede contraction, while the stiffness of the serial ECM must be high to transmit the forces to the load. Both the experimental results and the mechanistic model suggest that the combined stimulation through coordination reorients the ECM fibres in such a way that the parallel ECM stiffness is reduced, while the serial ECM stiffness is increased. In particular, 3 and 20 minutes of alternating electrical and mechanical stimulations increase the force by 18% and 31%, respectively.

Fabrication and characterization of optogenetic, multi-strip cardiac muscles.

Cardiac tissue engineering aims to recreate functional tissue constructs similar to the structure and function of the native myocardium. To date, in vitro tissue constructs lack the architectural complexity of a vascular network and the precise motor unit control of muscle fibers. Here, we present a method to construct engineered multi-strip cardiac muscle that simulates the bundle-like architecture of the native myocardium. Densely packed primary myocytes and cardiac fibroblasts were co-cultured with optogenetic, non-excitable cells. The resulting 3D syncytium triggered contraction upon localized blue light illumination to selectively activate and pace the multi-strip cardiac muscles, similar to the activity of pacemaker cells. Acting on a single load, we demonstrated graded force production through light-modulated multi-strip recruitment. These results demonstrate an in vitro platform of optogenetic, multi-strip cardiac muscles that can be used in a wide variety of applications, such as drug discovery, tissue engineering, and bio-hybrid robotic systems.