Predicting the preclinical efficacy of anti-fibrosis agents using a force-sensing fibrosis on chip system.

The high attrition rate of drug candidates contributes to the long duration and high cost in modern drug development. A major barrier in drug development is the poor predicting power of the preclinical models. In the current study, a human pulmonary fibrosis on chip system was developed for the preclinical evaluation of anti-fibrosis drugs. Pulmonary fibrosis is a severe disease characterized by progressive tissue stiffening that leads to respiration failure. To recapitulate the unique biomechanical feature of the fibrotic tissues, we developed flexible micropillars that can serve as in-situ force sensors to detect the changes in the mechanical properties of engineered lung microtissues. Using this system, we modeled the fibrogenesis of the alveolar tissues including the tissue stiffening and the expression of α-smooth muscle actin (α-SMA) and pro-collagen. Two anti-fibrosis drug candidates that are currently under clinical trials (KD025 and BMS-986020) were tested for their potential anti-fibrosis efficacy and the results were compared to those of FDA-approved anti-fibrosis drugs pirfenidone and nintedanib. Both pre-approval drugs were effective in inhibiting transforming growth factor beta 1 (TGF-ß1) induced increases in tissue contractile force, stiffness and expressions of fibrotic biomarkers, which are similar to the effects of FDA-approved anti-fibrosis drugs. These results demonstrated the potential utility of the force-sensing fibrosis on chip system in the pre-clinical development of anti-fibrosis drugs.

Fibrosis on a Chip for Screening of Anti-Fibrosis Drugs.

Idiopathic pulmonary fibrosis (IPF) is a chronic pathological disorder that targets alveoli interstitial tissues and is characterized by the progressive stiffening of alveolar membrane. The median survival rate of the patients with IPF is less than 5 years. Currently, IPF has no cure and there are few options to alleviate the progress of this disease. A critical roadblock in developing new anti-fibrosis therapies is the absence of reliable cell based in vitro models that can recapitulate the progressive features of this disease. Here a novel fibrotic microtissue on a chip system is created to model the fibrotic transition of the lung interstitial tissue and the effect of anti-fibrosis drugs on such transitions. This system will not only help to expedite the efficacy analysis of anti-fibrotic therapies but also help to unveil their potential mode of action.

Bioengineered Skeletal Muscle as a Model of Muscle Aging and Regeneration.

With age, adult skeletal muscle (SkM) is known to decrease in muscle mass, strength, and functional capacity, a state known as sarcopenia. Here we developed an in vitro three-dimensional (3D) bioengineered senescent SkM tissue using primary human myoblasts. These tissues exhibited the characteristics of atrophied muscle, including expression of senescent genes, decreased number of satellite cells, reduced number and size of myofibers, and compromised metabolism and calcium flux. As a result, senescent SkM tissues showed impaired ability to generate force in response to electrical stimulation compared with young tissues. Furthermore, in contrast to young SkM tissues, senescent tissues failed to regenerate in response to injury, possibly as a result of persistent apoptosis and failure to initiate a proliferation program. Our findings suggest that 3D senescent SkM may provide a powerful model for studying aging and a platform for drug testing and discovery of therapeutic compounds to improve the function of sarcopenic muscle. Impact statement Skeletal muscle (SkM) plays important physiological roles and has significant regenerative capacity. However, aged SkM lose their functionality and regeneration ability. In this article, we present a senescent human bioengineering SkM tissue model that can be used to investigate senescence, metabolic or genetic diseases that inflict SkM, and to test various strategies including novel small molecules that restore muscle function and promote regeneration. One key limitation of two-dimensional cell culture system is the detachment of contractile myotubes from the surface over time, thereby limiting the evaluation of myogenic function. Here we use primary human myoblasts, which exhibit all major hallmarks of aging to mimic the organization and function of native muscle. Using this system, we were able to measure the contractile function, calcium transients, and regeneration capacity of SkM tissues. We also evaluated the response of senescent SkM tissues to injury and their ability to regenerate and recover, compared with "young" tissues. Our results suggest that three-dimensional constructs enable organization of contractile units including myosin and actin filaments, thereby providing a powerful platform for the quantitative assessment of muscle myotubes in response to injury, genetic or metabolic disorders, or pharmacological testing.

Cyclic Stretching of Fibrotic Microtissue Array for Evaluation of Anti-Fibrosis Drugs.

INTRODUCTION: Progression of pulmonary fibrosis, characterized by the deterioration of lung tissue's mechanical properties, is affected by respiratory motion-induced dynamic loading. Since the development of anti-fibrosis drugs faces major hurdles in animal tests and human clinical trials, preclinical models that can recapitulate fibrosis progression under physiologically-relevant cyclic loading hold great promise. However, the integration of these two functions has not been achieved in existing models. METHODS: Recently we developed static human lung microtissue arrays that recapitulate the progressive changes in tissue mechanics during lung fibrogenesis. In the current study, we integrate the lung microtissue array with a membrane stretching system to enable dynamic loading to the microtissues. The effects of a pro-fibrotic agent and anti-fibrosis drugs were tested under cyclic stretching. RESULTS: Cyclic stretching that mimics respiratory motion was shown to affect the cytoskeletal organization and cellular orientation in the microtissue and cause the increase in microtissue contractility and stiffness. Fibrosis induction using TGF-ß1 further promoted fibrosis-related mechanical activity of the lung microtissues. Using this system, we examined the therapeutic effects of two FDA approved anti-fibrotic drugs. Our results showed that Nintedanib was able to fully inhibit TGF-ß1 induced force increase but only partially inhibited stretching induced force increase. In contrast, Pirfenidone was able to fully inhibit both TGF-ß1 induced force increase and stretching-induced force increase. CONCLUSIONS: Together, these results highlight the pathophysiologically-relevant modeling capability of the current fibrotic microtissue system and demonstrated the potential of this system to be used for anti-fibrosis drug screening.

Microclot array elastometry for integrated measurement of thrombus formation and clot biomechanics under fluid shear.

Blood clotting at the vascular injury site is a complex process that involves platelet adhesion and clot stiffening/contraction in the milieu of fluid flow. An integrated understanding of the hemodynamics and tissue mechanics regulating this process is currently lacking due to the absence of an experimental system that can simultaneously model clot formation and measure clot mechanics under shear flow. Here we develop a microfluidic-integrated microclot-array-elastometry system (clotMAT) that recapitulates dynamic changes in clot mechanics under physiological shear. Treatments with procoagulants and platelet antagonists and studies with diseased patient plasma demonstrate the ability of the system to assay clot biomechanics associated with common antiplatelet treatments and bleeding disorders. The changes of clot mechanics under biochemical treatments and shear flow demonstrate independent yet equally strong effects of these two stimulants on clot stiffening. This microtissue force sensing system may have future research and diagnostic potential for various bleeding disorders.

Fibrotic microtissue array to predict anti-fibrosis drug efficacy.

Fibrosis is a severe health problem characterized by progressive stiffening of tissues which causes organ malfunction and failure. A major bottleneck in developing new anti-fibrosis therapies is the lack of in vitro models that recapitulate dynamic changes in tissue mechanics during fibrogenesis. Here we create membranous human lung microtissues to model key biomechanical events occurred during lung fibrogenesis including progressive stiffening and contraction of alveolar tissue, decline in alveolar tissue compliance and traction force-induced bronchial dilation. With these capabilities, we provide proof of principle for using this fibrotic tissue array for multi-parameter, phenotypic analysis of the therapeutic efficacy of two anti-fibrosis drugs recently approved by the FDA. Preventative treatments with Pirfenidone and Nintedanib reduce tissue contractility and prevent tissue stiffening and decline in tissue compliance. In a therapeutic treatment regimen, both drugs restore tissue compliance. These results highlight the pathophysiologically relevant modeling capability of our novel fibrotic microtissue system.

Tempo-Spatial Compressed Sensing of Organ-on-a-Chip for Pervasive Health.

As a micro-engineered biomimetic system to replicate key functions of living organs, organ-on-a-chip (OC) technology provides a high-throughput model for investigating complex cell interactions with both high temporal and spatial resolutions in biological studies. Typically, microscopy and high-speed video cameras are used for data acquisition, which are expensive and bulky. Recently, compressed sensing (CS) has increasingly attracted attentions due to its extremely low-complexity structure and low sampling rate. However, there is no CS solution tailored for tempo-spatial information acquisition. In this paper, we propose tempo-spatial CS (TS-CS), a unified CS architecture for OC stream, which achieves significant cost reduction and truly combines sensing with compression along the temporal and spatial domains. We point out that TS-CS can consistently achieve better performance by exploiting tempo-spatial compressibility in OC data. To this end, we comprehensively evaluate the system performance by employing four different bases for CS. With comparison to the traditional way, we show that TS-CS always obtains better recovery result with a throughput bound and can achieve around throughput improvement under a reconstruction demand by applying discrete cosine transform matrix as the basis.

NANOG restores the impaired myogenic differentiation potential of skeletal myoblasts after multiple population doublings.

Adult skeletal muscle regeneration relies on the activity of satellite cells residing in the skeletal muscle niche. However, systemic and intrinsic factors decrease the myogenic differentiation potential of satellite cells thereby impairing muscle regeneration. Here we present data showing that late passage C2C12 myoblasts exhibited significantly impaired myogenic differentiation potential that was accompanied by impaired expression of myogenic regulatory factors (Myf5, MyoD, Myogenin, and MRF4) and members of myocyte enhancer factor 2 family. Notably, ectopic expression of NANOG preserved the morphology and restored the myogenic differentiation capacity of late passage myoblasts, possibly by restoring the expression level of these myogenic factors. Muscle regeneration was effective in 2D cultures and in 3D skeletal microtissues mimicking the skeletal muscle niche. The presence of NANOG was required for at least 15days to reverse the impaired differentiation potential of myoblasts. However, it was critical to remove NANOG during the process of maturation, as it inhibited myotube formation. Finally, myoblasts that were primed by NANOG maintained their differentiation capacity for 20days after NANOG withdrawal, suggesting potential epigenetic changes. In conclusion, these results shed light on the potential of NANOG to restore the myogenic differentiation potential of myoblasts, which is impaired after multiple rounds of cellular division, and to reverse the loss of muscle regeneration.

NANOG Restores Contractility of Mesenchymal Stem Cell-Based Senescent Microtissues.

Mesenchymal stem cells (MSCs) have been extensively used in the field of tissue engineering as a source of smooth muscle cells (SMCs). However, recent studies showed deficits in the contractile function of SMCs derived from senescent MSCs and there are no available strategies to restore the contractile function that is impaired due to cellular or organismal senescence. In this study, we developed a tetracycline-regulatable system and employed micropost tissue arrays to evaluate the effects of the embryonic transcription factor, NANOG, on the contractility of senescent MSCs. Using this system, we show that expression of NANOG fortified the actin cytoskeleton and restored contractile function that was impaired in senescent MSCs. NANOG increased the expression of smooth muscle α-actin (ACTA2) as well as the contractile force generated by cells in three-dimensional microtissues. Interestingly, NANOG worked together with transforming growth factor-beta1 to further enhance the contractility of senescent microtissues. The effect of NANOG on contractile function was sustained for about 10 days after termination of its expression. Our results show that NANOG could reverse the effects of stem cell senescence and restore the myogenic differentiation potential of senescent MSCs. These findings may enable development of novel strategies to restore the function of senescent cardiovascular and other SMC-containing tissues.

NANOG Reverses the Myogenic Differentiation Potential of Senescent Stem Cells by Restoring ACTIN Filamentous Organization and SRF-Dependent Gene Expression.

Cellular senescence as a result of organismal aging or progeroid diseases leads to stem cell pool exhaustion hindering tissue regeneration and contributing to the progression of age related disorders. Here we discovered that ectopic expression of the pluripotent factor NANOG in senescent or progeroid myogenic progenitors reversed cellular aging and restored completely the ability to generate contractile force. To elicit its effects, NANOG enabled reactivation of the ROCK and Transforming Growth Factor (TGF)-ß pathways-both of which were impaired in senescent cells-leading to ACTIN polymerization, MRTF-A translocation into the nucleus and serum response factor (SRF)-dependent myogenic gene expression. Collectively our data reveal that cellular senescence can be reversed and provide a novel strategy to regain the lost function of aged stem cells without reprogramming to the pluripotent state. Stem Cells 2017;35:207-221.

Lung Microtissue Array to Screen the Fibrogenic Potential of Carbon Nanotubes.

Due to their excellent physical and chemical characteristics, multi-wall carbon nanotubes (MWCNT) have the potential to be used in structural composites, conductive materials, sensors, drug delivery and medical imaging. However, because of their small-size and light-weight, the applications of MWCNT also raise health concerns. In vivo animal studies have shown that MWCNT cause biomechanical and genetic alterations in the lung tissue which lead to lung fibrosis. To screen the fibrogenic risk factor of specific types of MWCNT, we developed a human lung microtissue array device that allows real-time and in-situ readout of the biomechanical properties of the engineered lung microtissue upon MWCNT insult. We showed that the higher the MWCNT concentration, the more severe cytotoxicity was observed. More importantly, short type MWCNT at low concentration of 50 ng/ml stimulated microtissue formation and contraction force generation, and caused substantial increase in the fibrogenic marker miR-21 expression, indicating the high fibrogenic potential of this specific carbon nanotube type and concentration. The presented microtissue array system provides a powerful tool for high-throughput examination of the therapeutic and toxicological effects of target compounds in realistic tissue environment.

YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells.

Myelination is essential for nervous system function. Schwann cells interact with neurons and the basal lamina to myelinate axons using known receptors, signals and transcription factors. In contrast, the transcriptional control of axonal sorting and the role of mechanotransduction in myelination are largely unknown. Yap and Taz are effectors of the Hippo pathway that integrate chemical and mechanical signals in cells. We describe a previously unknown role for the Hippo pathway in myelination. Using conditional mutagenesis in mice, we show that Taz is required in Schwann cells for radial sorting and myelination and that Yap is redundant with Taz. Yap and Taz are activated in Schwann cells by mechanical stimuli and regulate Schwann cell proliferation and transcription of basal lamina receptor genes, both necessary for radial sorting of axons and subsequent myelination. These data link transcriptional effectors of the Hippo pathway and of mechanotransduction to myelin formation in Schwann cells.