Directed differentiation of human iPSCs into mesenchymal lineages by optogenetic control of TGF-ß signaling.

In tissue development and homeostasis, transforming growth factor (TGF)-ß signaling is finely coordinated by latent forms and matrix sequestration. Optogenetics can offer precise and dynamic control of cell signaling. We report the development of an optogenetic human induced pluripotent stem cell system for TGF-ß signaling and demonstrate its utility in directing differentiation into the smooth muscle, tenogenic, and chondrogenic lineages. Light-activated TGF-ß signaling resulted in expression of differentiation markers at levels close to those in soluble factor-treated cultures, with minimal phototoxicity. In a cartilage-bone model, light-patterned TGF-ß gradients allowed the establishment of hyaline-like layer of cartilage tissue at the articular surface while attenuating with depth to enable hypertrophic induction at the osteochondral interface. By selectively activating TGF-ß signaling in co-cultures of light-responsive and non-responsive cells, undifferentiated and differentiated cells were simultaneously maintained in a single culture with shared medium. This platform can enable patient-specific and spatiotemporally precise studies of cellular decision making.

A multi-organ chip with matured tissue niches linked by vascular flow.

Engineered tissues can be used to model human pathophysiology and test the efficacy and safety of drugs. Yet, to model whole-body physiology and systemic diseases, engineered tissues with preserved phenotypes need to physiologically communicate. Here we report the development and applicability of a tissue-chip system in which matured human heart, liver, bone and skin tissue niches are linked by recirculating vascular flow to allow for the recapitulation of interdependent organ functions. Each tissue is cultured in its own optimized environment and is separated from the common vascular flow by a selectively permeable endothelial barrier. The interlinked tissues maintained their molecular, structural and functional phenotypes over 4 weeks of culture, recapitulated the pharmacokinetic and pharmacodynamic profiles of doxorubicin in humans, allowed for the identification of early miRNA biomarkers of cardiotoxicity, and increased the predictive values of clinically observed miRNA responses relative to tissues cultured in isolation and to fluidically interlinked tissues in the absence of endothelial barriers. Vascularly linked and phenotypically stable matured human tissues may facilitate the clinical applicability of tissue chips.

milliPillar: A Platform for the Generation and Real-Time Assessment of Human Engineered Cardiac Tissues.

Engineered cardiac tissues derived from human induced pluripotent stem cells (iPSCs) are increasingly used for drug discovery, pharmacology and in models of development and disease. While there are numerous platforms to engineer cardiac tissues, they often require expensive and nonconventional equipment and utilize complex video-processing algorithms. As a result, only specialized academic laboratories have been able to harness this technology. In addition, methodologies and tissue features have been challenging to reproduce between different groups and models. Here, we describe a facile technology (milliPillar) that covers the entire pipeline required for studies of engineered cardiac tissues. We include methodologies for (i) platform fabrication, (ii) cardiac tissue generation, (iii) electrical stimulation, (iv) automated real-time data acquisition, and (v) advanced video analyses. We validate these methodologies and demonstrate the versatility of the platform by showcasing the fabrication of tissues in different hydrogel materials and using cardiomyocytes derived from different iPSC lines in combination with different types of stromal cells. We also validate the long-term culture of tissues within the platform and provide protocols for automated analysis of force generation and calcium flux using both brightfield and fluorescence imaging. Lastly, we demonstrate the compatibility of the milliPillar platform with electromechanical stimulation to enhance cardiac tissue function. We expect that this resource will provide a valuable and user-friendly tool for the generation and real-time assessment of engineered human cardiac tissues for basic and translational studies.

Bioengineered optogenetic model of human neuromuscular junction.

Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function.

Tissue engineered autologous cartilage-bone grafts for temporomandibular joint regeneration.

Joint disorders can be detrimental to quality of life. There is an unmet need for precise functional reconstruction of native-like cartilage and bone tissues in the craniofacial space and particularly for the temporomandibular joint (TMJ). Current surgical methods suffer from lack of precision and comorbidities and frequently involve multiple operations. Studies have sought to improve craniofacial bone grafts without addressing the cartilage, which is essential to TMJ function. For the human-sized TMJ in the Yucatan minipig model, we engineered autologous, biologically, and anatomically matched cartilage-bone grafts for repairing the ramus-condyle unit (RCU), a geometrically intricate structure subjected to complex loading forces. Using image-guided micromilling, anatomically precise scaffolds were created from decellularized bone matrix and infused with autologous adipose-derived chondrogenic and osteogenic progenitor cells. The resulting constructs were cultured in a dual perfusion bioreactor for 5 weeks before implantation. Six months after implantation, the bioengineered RCUs maintained their predefined anatomical structure and regenerated full-thickness, stratified, and mechanically robust cartilage over the underlying bone, to a greater extent than either autologous bone-only engineered grafts or acellular scaffolds. Tracking of implanted cells and parallel bioreactor studies enabled additional insights into the progression of cartilage and bone regeneration. This study demonstrates the feasibility of TMJ regeneration using anatomically precise, autologous, living cartilage-bone grafts for functional, personalized total joint replacement. Inclusion of the adjacent tissues such as soft connective tissues and the TMJ disc could further extend the functional integration of engineered RCUs with the host.

Integrated human organ-on-a-chip model for predictive studies of anti-tumor drug efficacy and cardiac safety.

Traditional drug screening models are often unable to faithfully recapitulate human physiology in health and disease, motivating the development of microfluidic organs-on-a-chip (OOC) platforms that can mimic many aspects of human physiology and in the process alleviate many of the discrepancies between preclinical studies and clinical trials outcomes. Linsitinib, a novel anti-cancer drug, showed promising results in pre-clinical models of Ewing Sarcoma (ES), where it suppressed tumor growth. However, a Phase II clinical trial in several European centers with patients showed relapsed and/or refractory ES. We report an integrated, open setting, imaging and sampling accessible, polysulfone-based platform, featuring minimal hydrophobic compound binding. Two bioengineered human tissues - bone ES tumor and heart muscle - were cultured either in isolation or in the integrated platform and subjected to a clinically used linsitinib dosage. The measured anti-tumor efficacy and cardiotoxicity were compared with the results observed in the clinical trial. Only the engineered tumor tissues, and not monolayers, recapitulated the bone microenvironment pathways targeted by linsitinib, and the clinically-relevant differences in drug responses between non-metastatic and metastatic ES tumors. The responses of non-metastatic ES tumor tissues and heart muscle to linsitinib were much closer to those observed in the clinical trial for tissues cultured in an integrated setting than for tissues cultured in isolation. Drug treatment of isolated tissues resulted in significant decreases in tumor viability and cardiac function. Meanwhile, drug treatment in an integrated setting showed poor tumor response and less cardiotoxicity, which matched the results of the clinical trial. Overall, the integration of engineered human tumor and cardiac tissues in the integrated platform improved the predictive accuracy for both the direct and off-target effects of linsitinib. The proposed approach could be readily extended to other drugs and tissue systems.

Engineering of human cardiac muscle electromechanically matured to an adult-like phenotype.

The application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to ß-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.

Author Correction: Advanced maturation of human cardiac tissue grown from pluripotent stem cells.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Novel disposable cell block processing device and method for high cellular yield.

BACKGROUND: Cell blocks represent one of several cytology preparations, and play a significant role in providing tissue for ancillary studies, including immunohistochemistry and molecular testing. To the authors' knowledge, there currently is no standardized cell block processing method, and cellular yield often is suboptimal. To overcome the shortcomings of available methods, the authors developed a novel disposable cell block device, XCellent, that uses centrifugation to collect cells into a sample chamber. The sample chamber contains and protects the specimen during tissue processing and also is embedded and sectioned. The primary objectives of the current study were to compare cellular yield, cytomorphological detail, and processing times of HistoGel and XCellent. METHODS: Split sample testing of primarily fluid specimens was performed. Group 1 comprised large-volume and/or cloudy fluids and group 2 was residual samples in PreservCyt solution. Two cell blocks, 1 HistoGel cell block and 1 XCellent cell block, were processed from each sample. Cellularity was assessed using computerized image analysis and cytomorphology from 1+ to 3+. Processing times for 16 cell blocks also were recorded. RESULTS: A total of 50 cell blocks (25 HistoGel samples and 25 XCellent samples) were prepared. On average, cell blocks prepared using XCellent were 371% more cellular than those prepared with HistoGel and had better cytomorphology. Less preparation time was required for bulk processing of XCellent cell blocks (11 minutes) compared with the same quantity of bulk processed using HistoGel (38 minutes). CONCLUSIONS: XCellent provides an alternative cell block processing technique that offers high cellular yield and cytomorphology. In addition, the method uses the standard centrifugation process, which integrates into the existing cytology laboratory workflow.

A Multimaterial Microphysiological Platform Enabled by Rapid Casting of Elastic Microwires.

Due to escalating drug developmental costs and limitations of cardiotoxicity screening, there is an urgent need to develop robust in vitro 3D tissue culture platforms that can both facilitate the culture of human cardiac tissues and provide noninvasive functional readouts predictive of cardiotoxicity in clinical settings. However, such platforms commonly require complex fabrication procedures that are difficult to scale up to high-throughput testing platforms. Here, innovative multimaterial processing into a scalable and functional platform is proposed in the format of a 96-well plate. Three classes of materials are integrated into the platform. An array of soft elastic microwires is used both as anchors for tissue formation as well as sensors for recording tissue contraction. Conductive carbon electrodes are embedded into the plate to drive electrical stimulation for tissue maturation and pace tissue contraction during drug testing. The bulk of the device is made of rigid polystyrene plastic to eliminate drug-absorbing polydimethylsiloxane (PDMS). The platform has higher throughput than the current state-of-the-art devices, at a significantly reduced cost of manufacturing and tissue production.

Advanced maturation of human cardiac tissue grown from pluripotent stem cells.

Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1-6. However, the predictive power of these models is presently limited by the immature state of the cells1, 2, 5, 6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force-frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.

Human bone perivascular niche-on-a-chip for studying metastatic colonization.

Eight out of 10 breast cancer patients die within 5 years after the primary tumor has spread to the bones. Tumor cells disseminated from the breast roam the vasculature, colonizing perivascular niches around blood capillaries. Slow flows support the niche maintenance by driving the oxygen, nutrients, and signaling factors from the blood into the interstitial tissue, while extracellular matrix, endothelial cells, and mesenchymal stem cells regulate metastatic homing. Here, we show the feasibility of developing a perfused bone perivascular niche-on-a-chip to investigate the progression and drug resistance of breast cancer cells colonizing the bone. The model is a functional human triculture with stable vascular networks within a 3D native bone matrix cultured on a microfluidic chip. Providing the niche-on-a-chip with controlled flow velocities, shear stresses, and oxygen gradients, we established a long-lasting, self-assembled vascular network without supplementation of angiogenic factors. We further show that human bone marrow-derived mesenchymal stem cells, which have undergone phenotypical transition toward perivascular cell lineages, support the formation of capillary-like structures lining the vascular lumen. Finally, breast cancer cells exposed to interstitial flow within the bone perivascular niche-on-a-chip persist in a slow-proliferative state associated with increased drug resistance. We propose that the bone perivascular niche-on-a-chip with interstitial flow promotes the formation of stable vasculature and mediates cancer cell colonization.

Tissue-engineered autologous grafts for facial bone reconstruction.

Facial deformities require precise reconstruction of the appearance and function of the original tissue. The current standard of care-the use of bone harvested from another region in the body-has major limitations, including pain and comorbidities associated with surgery. We have engineered one of the most geometrically complex facial bones by using autologous stromal/stem cells, native bovine bone matrix, and a perfusion bioreactor for the growth and transport of living grafts, without bone morphogenetic proteins. The ramus-condyle unit, the most eminent load-bearing bone in the skull, was reconstructed using an image-guided personalized approach in skeletally mature Yucatán minipigs (human-scale preclinical model). We used clinically approved decellularized bovine trabecular bone as a scaffolding material and crafted it into an anatomically correct shape using image-guided micromilling to fit the defect. Autologous adipose-derived stromal/stem cells were seeded into the scaffold and cultured in perfusion for 3 weeks in a specialized bioreactor to form immature bone tissue. Six months after implantation, the engineered grafts maintained their anatomical structure, integrated with native tissues, and generated greater volume of new bone and greater vascular infiltration than either nonseeded anatomical scaffolds or untreated defects. This translational study demonstrates feasibility of facial bone reconstruction using autologous, anatomically shaped, living grafts formed in vitro, and presents a platform for personalized bone tissue engineering.

Introducer Development for Coronary Sinus Access From Parasternal Mediastinotomy.

OBJECTIVE: Right parasternal mediastinotomy with right atriotomy has been used clinically for pacemaker insertion. A similar approach might facilitate access to the coronary sinus for biventricular pacing and other manipulations when more conventional approaches are not feasible. The primary barrier to this is lack of appropriate introducers and techniques. METHODS: Anatomically derived introducers were developed in 2 anesthetized domestic pigs using data from computerized axial thoracic tomography. Each digitized tomogram defined a unique introducer shape and was constructed using 3-dimensional (3D) modeling software and printing. Each parent pig then underwent surgery demonstrating coronary sinus lead insertion, using its custom-configured introducer. Next, with institutional review board approval, 65 patients were identified who had undergone conventional endocardial coronary sinus lead insertion followed by thoracic scanning. These tomograms were used to design appropriately curved introducers for human anatomy. RESULTS: Fifty-one introducer paths were defined following anatomic pathways and avoiding bends inconsistent with materials used for commercial peel-away introducers. Each path was defined by a bend and distance toward the coronary sinus ostium and a hook and twist out of plane to align with the local orientation of the coronary sinus. The average dimensions were the following: distance, 67 mm; bend angle, 47 degrees; hook angle, 39 degrees; and twist angle, 20 degrees. A prototype cannula was tested for fit in a fresh frozen postmortem human specimen. CONCLUSIONS: Parasternal mediastinotomy access to the coronary sinus for cardiac resynchronization, mitral annuloplasty, and instrumentation is feasible. Human computerized tomographic scans can be used to define curvatures and dimensions for marketed introducers.

Combined effects of chemical priming and mechanical stimulation on mesenchymal stem cell differentiation on nanofiber scaffolds.

Functional tissue engineering of connective tissues such as the anterior cruciate ligament (ACL) remains a significant clinical challenge, largely due to the need for mechanically competent scaffold systems for grafting, as well as a reliable cell source for tissue formation. We have designed an aligned, polylactide-co-glycolide (PLGA) nanofiber-based scaffold with physiologically relevant mechanical properties for ligament regeneration. The objective of this study is to identify optimal tissue engineering strategies for fibroblastic induction of human mesenchymal stem cells (hMSC), testing the hypothesis that basic fibroblast growth factor (bFGF) priming coupled with tensile loading will enhance hMSC-mediated ligament regeneration. It was observed that compared to the unloaded, as well as growth factor-primed but unloaded controls, bFGF stimulation followed by physiologically relevant tensile loading enhanced hMSC proliferation, collagen production and subsequent differentiation into ligament fibroblast-like cells, upregulating the expression of types I and III collagen, as well as tenasin-C and tenomodulin. The results of this study suggest that bFGF priming increases cell proliferation, while mechanical stimulation of the hMSCs on the aligned nanofiber scaffold promotes fibroblastic induction of these cells. In addition to demonstrating the potential of nanofiber scaffolds for hMSC-mediated functional ligament tissue engineering, this study yields new insights into the interactive effects of chemical and mechanical stimuli on stem cell differentiation.

Bioreactor cultivation of anatomically shaped human bone grafts.

In this chapter, we describe a method for engineering bone grafts in vitro with the specific geometry of the temporomandibular joint (TMJ) condyle. The anatomical geometry of the bone grafts was segmented from computed tomography (CT) scans, converted to G-code, and used to machine decellularized trabecular bone scaffolds into the identical shape of the condyle. These scaffolds were seeded with human bone marrow-derived mesenchymal stem cells (MSCs) using spinner flasks and cultivated for up to 5 weeks in vitro using a custom-designed perfusion bioreactor system. The flow patterns through the complex geometry were modeled using the FloWorks module of SolidWorks to optimize bioreactor design. The perfused scaffolds exhibited significantly higher cellular content, better matrix production, and increased bone mineral deposition relative to non-perfused (static) controls after 5 weeks of in vitro cultivation. This technology is broadly applicable for creating patient-specific bone grafts of varying shapes and sizes.

Microfluidic bioreactor for dynamic regulation of early mesodermal commitment in human pluripotent stem cells.

During development and regeneration, tissues emerge from coordinated sequences of stem cell renewal, specialization and assembly that are orchestrated by cascades of regulatory signals. The complex and dynamic in vivo milieu cannot be replicated using standard in vitro techniques. Microscale technologies now offer potential for conducting highly controllable and sophisticated experiments at biologically relevant scales, with real-time insights into cellular responses. We developed a microbioreactor providing time sequences of space-resolved gradients of multiple molecular factors in three-dimensional (3D) cell culture settings, along with a versatile, high-throughput operation and imaging compatibility. A single microbioreactor yields up to 120 data points, corresponding to 15 replicates of a gradient with 8 concentration levels. Embryoid bodies (EBs) obtained from human embryonic and induced pluripotent stem cells (hESC, hiPSC) were exposed to concentration gradients of Wnt3a, Activin A, BMP4 and their inhibitors, to get new insights into the early-stage fate specification and mesodermal lineage commitment. We were able to evaluate the initiation of mesodermal induction by measuring and correlating the gene expression profiles to the concentration gradients of mesoderm-inducing morphogens. We propose that the microbioreactor systems combining spatial and temporal gradients of molecular and physical factors to hESC and hiPSC cultures can form a basis for predictable in vitro models of development and disease.

Biomimetic perfusion and electrical stimulation applied in concert improved the assembly of engineered cardiac tissue.

Maintenance of normal myocardial function depends intimately on synchronous tissue contraction, driven by electrical activation and on adequate nutrient perfusion in support thereof. Bioreactors have been used to mimic aspects of these factors in vitro to engineer cardiac tissue but, due to design limitations, previous bioreactor systems have yet to simultaneously support nutrient perfusion, electrical stimulation and unconstrained (i.e. not isometric) tissue contraction. To the best of our knowledge, the bioreactor system described herein is the first to integrate these three key factors in concert. We present the design of our bioreactor and characterize its capability in integrated experimental and mathematical modelling studies. We then cultured cardiac cells obtained from neonatal rats in porous, channelled elastomer scaffolds with the simultaneous application of perfusion and electrical stimulation, with controls excluding either one or both of these two conditions. After 8 days of culture, constructs grown with simultaneous perfusion and electrical stimulation exhibited substantially improved functional properties, as evidenced by a significant increase in contraction amplitude (0.23 ± 0.10% vs 0.14 ± 0.05%, 0.13 ± 0.08% or 0.09 ± 0.02% in control constructs grown without stimulation, without perfusion, or either stimulation or perfusion, respectively). Consistently, these constructs had significantly improved DNA contents, cell distribution throughout the scaffold thickness, cardiac protein expression, cell morphology and overall tissue organization compared to control groups. Thus, the simultaneous application of medium perfusion and electrical conditioning enabled by the use of the novel bioreactor system may accelerate the generation of fully functional, clinically sized cardiac tissue constructs.

Engineering anatomically shaped human bone grafts.

The ability to engineer anatomically correct pieces of viable and functional human bone would have tremendous potential for bone reconstructions after congenital defects, cancer resections, and trauma. We report that clinically sized, anatomically shaped, viable human bone grafts can be engineered by using human mesenchymal stem cells (hMSCs) and a "biomimetic" scaffold-bioreactor system. We selected the temporomandibular joint (TMJ) condylar bone as our tissue model, because of its clinical importance and the challenges associated with its complex shape. Anatomically shaped scaffolds were generated from fully decellularized trabecular bone by using digitized clinical images, seeded with hMSCs, and cultured with interstitial flow of culture medium. A bioreactor with a chamber in the exact shape of a human TMJ was designed for controllable perfusion throughout the engineered construct. By 5 weeks of cultivation, tissue growth was evidenced by the formation of confluent layers of lamellar bone (by scanning electron microscopy), markedly increased volume of mineralized matrix (by quantitative microcomputer tomography), and the formation of osteoids (histologically). Within bone grafts of this size and complexity cells were fully viable at a physiologic density, likely an important factor of graft function. Moreover, the density and architecture of bone matrix correlated with the intensity and pattern of the interstitial flow, as determined in experimental and modeling studies. This approach has potential to overcome a critical hurdle-in vitro cultivation of viable bone grafts of complex geometries-to provide patient-specific bone grafts for craniofacial and orthopedic reconstructions.

Design and evaluation of a hybrid passive and active gravity neutral orthosis (GNO).

Neuromuscular diseases (NMD), including Spinal Muscular Atrophy (SMA) and Duchenne Muscular Dystrophy (DMD), result in progressive muscular weakness that often leaves patients functionally dependent on caregivers for many activities of daily living (ADL) such as eating, bathing, grooming (touching the face and head), reaching (grabbing for objects), and dressing. In severe cases, patients are unable to perform even the simplest of activities from exploring their 3D space to touching their own face. The ability to move and initiate age appropriate tasks, such as playing and exploration, are considered to be of vital importance to both their physical and cognitive development. Therefore, to improve quality of life and reduce dependence on caregivers in children and young adults with NMD, we designed, built and evaluated an assistive, active orthosis to support arm function. The goal of this project is the development and evaluation of a mechanical arm orthosis to both encourage and assist functional arm movement while providing the user a sense of independence and control over one's own body.