Restoring anatomical complexity of a left ventricle wall as a step toward bioengineering a human heart with human induced pluripotent stem cell-derived cardiac cells.

The heart is a highly complex, multicellular solid organ with energy-demanding processes that require a dense vascular network, extensive cell-cell interactions, and extracellular matrix (ECM)-mediated crosstalk among heterogeneous cell populations. Here, we describe the regeneration of left ventricular (LV) wall using decellularized whole rabbit heart scaffolds recellularized exclusively with human induced pluripotent stem cell-derived endothelial cells, cardiomyocytes, and other cardiac cell types. Cells were sequentially delivered to the scaffold using an optimized endothelial cell:cardiomyocyte media. Macroscopic assessment after 60 days showed that the LV wall of recellularized hearts was anatomically restored to full thickness from base to apex and endocardium to epicardium. Histologic analysis of the recellularized LV wall revealed a heterogeneous pool of cardiac cells containing aligned cardiac troponin T-positive cells in close contact with ECM; vessels varied from large artery-like, surrounded by smooth muscle actin+ cells, to capillary-like. Vessel patency was demonstrated after perfusion of recellularized hearts transplanted into the femoral artery bed of a pig. The construct exhibited visible beating and responded to chronotropic drug administration. These results demonstrate the ability to tissue engineer a vascularized, full-thickness LV wall with an unparalleled level of microanatomical organization and multicellular composition, using decellularized ECM and human cardiomyocytes, endothelial cells, and other cardiac cell types. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrix (ECM) is a bioactive template for tissue engineering, but recellularizing acellular whole heart scaffolds is challenging. Here, we successfully revascularized and repopulated a large, full-thickness portion of a ventricle using human induced pluripotent stem cell-derived endothelial and cardiac cells. At 60 days, histologic studies showed that the microanatomical organization and cellular composition of this region was similar to that of the native heart. The recellularized heart showed visible beating and responded appropriately to heartbeat-altering drugs. Vessels surrounded by smooth muscle cells and endothelial cells supported blood flow through the vessels of a recellularized heart that was surgically connected to a pig femoral artery. These findings move this approach closer to the possibility of clinical translation.

Population-Based Study on the All-Cause and Cause-Specific Risks of Mortality among Long-Term Opioid Analgesics Users without Cancer in Taiwan.

(1) Background: The prevalence of opioid use in Taiwan increased by 41% between 2002 and 2014. However, little is known regarding the risk of mortality among long-term opioid analgesics users who do not have cancer. This study investigated this mortality risk with an emphasis on the calendar year and patients' age and sex. (2) Methods: This retrospective cohort study included 12,990 adult individuals without cancer who were long-term users of opioid analgesics and were randomly selected from the data set of Taiwan's National Health Insurance program from 2000 to 2012. They were then followed up through 2013. Information on the underlying causes of death was retrieved from the Taiwan Death Registry. Age, sex, and calendar year-standardized mortality ratios (SMRs) of all-cause and cause-specific mortality were calculated with reference to those of the general population. (3) Results: With up to 14 years of follow-up, 558 individuals had all-cause mortality in 48,020 person-years (cumulative mortality: 4.3%, mortality rate: 11.62 per 1000 person-years). Compared with the general population, the all-cause SMR of 4.30 (95% confidence interval (95% CI): 3.95-4.66) was significantly higher: it was higher in men than in women, declined with calendar year and age, and was significantly higher for both natural (4.15, 95% CI: 3.78-4.53) and unnatural (5.04, 95% CI: 3.88-6.45) causes. (4) Conclusions: Long-term opioid analgesics use among individuals without cancer in Taiwan was associated with a significantly increased risk of mortality. The notably increased mortality in younger adults warrants attention. Strategies to reduce long-term opioid analgesics use, especially their overuse or misuse, are in an urgent need.

Cues from human atrial extracellular matrix enrich the atrial differentiation of human induced pluripotent stem cell-derived cardiomyocytes.

New robust and reproducible differentiation approaches are needed to generate induced pluripotent stem cell (iPSC)-derived cardiomyocytes of specific subtypes in predictable quantities for tissue-specific disease modeling, tissue engineering, and eventual clinical translation. Here, we assessed whether powdered decellularized extracellular matrix (dECM) particles contained chamber-specific cues that could direct the cardiac differentiation of human iPSCs toward an atrial phenotype. Human hearts were dissected and the left ventricle (LV) and left atria (LA) were isolated, minced, and decellularized using an adapted submersion decellularization technique to generate chamber-specific powdered dECM. Comparative proteomic analyses showed chamber-specific dECM segregation, with atrial- and ventricle-specific proteins uniquely present in powdered dECM-hA and dECM-hV, respectively. Cell populations differentiated in the presence of dECM-hA showed upregulated atrial molecular markers and a two-fold increase in the number of atrial-like cells as compared with cells differentiated with dECM-hV or no dECM (control). Finally, electrophysiological data showed an increase in action potentials characteristic of atrial-like cells in the dECM-hA group. These findings support the hypothesis that dECM powder derived from human atria retained endogenous cues to drive cardiac differentiation toward an atrial fate.

Gelatin Promotes Cell Retention Within Decellularized Heart Extracellular Matrix Vasculature and Parenchyma.

INTRODUCTION: Recellularization of organ decellularized extracellular matrix (dECM) offers a potential solution for organ shortage in allograft transplantation. Cell retention rates have ranged from 10 to 54% in varying approaches for reseeding cells in whole organ dECM scaffolds. We aimed to improve recellularization by using soluble gelatin as a cell carrier to deliver endothelial cells to the coronary vasculature and cardiomyocytes to the parenchyma in a whole decellularized rat heart. METHODS: Rat aortic endothelial cells (RAECs) were perfused over decellularized porcine aorta in low (1%) and high (5%) concentrations of gelatin to assess attachment to a vascular dECM model. After establishing cell viability and proliferation in 1% gelatin, we used 1% gelatin as a carrier to deliver RAECs and neonatal rat cardiomyocytes (NRCMs) to decellularized adult rat hearts. Immediate cell retention in the matrix was quantified, and recellularized hearts were evaluated for visible contractions up to 35 days after recellularization. RESULTS: We demonstrated that gelatin increased RAEC attachment to decellularized porcine aorta; blocking integrin receptors reversed this effect. In the whole rat heart gelatin (1%) increased retention of both RAECs and NRCMs respectively, compared with the control group (no gelatin). Gelatin was associated with visible contractions of NRCMs within hearts (87% with gelatin vs. 13% control). CONCLUSIONS: Gelatin was an effective cell carrier for increasing cell retention and contraction in dECM. The gelatin-cell-ECM interactions likely mediated by integrin.

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation.

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing "cellularization" at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more "physiological" method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

Inverted orientation improves decellularization of whole porcine hearts.

In structurally heterogeneous organs, such as heart, it is challenging to retain extracellular matrix integrity in the thinnest regions (eg, valves) during perfusion decellularization and completely remove cellular debris from thicker areas. The high inflow rates necessary to maintain physiologic pressure can distend or damage thin tissues, but lower pressures prolong the process and increase the likelihood of contamination. We examined two novel retrograde decellularization methods for porcine hearts: inverting the heart or venting the apex to decrease inflow rate. We measured flow dynamics through the aorta (Ao) and pulmonary artery (PA) at different Ao pressures and assessed the heart's appearance, turbidity of the outflow solutions, and coronary perfusion efficiency. We used rectangle image fitting of decellularized heart images to obtain a heart shape index. Using nonlinear optical microscopy, we determined the microstructure of collagen and elastin fibers of the aortic valve cusps. DNA, glycosaminoglycan, and residual detergent levels were compared. The inverted method was superior to the vented method, as shown by a higher coronary perfusion efficiency, more cell debris outflow, higher collagen and elastin content inside the aortic valve, lower DNA content, and better retention of the heart shape after decellularization. To our knowledge, this is the first study to use flow dynamics in a whole heart throughout the decellularization procedure to provide real-time information about the success of the process and the integrity of the vulnerable regions of the matrix. Heart orientation was important in optimizing decellularization efficiency and maintaining extracellular matrix integrity. STATEMENT OF SIGNIFICANCE: The use of decellularized tissue as a suitable scaffold for engineered tissue has emerged over the past decade as one of the most promising biofabrication platforms. The decellularization process removes all native cells, leaving the natural biopolymers, extracellular matrix materials and native architecture intact. This manuscript describes heart orientation as important in optimizing decellularization efficiency and maintaining extracellular matrix integrity. To our knowledge, this is the first study to assess flow dynamics in a whole heart throughout the decellularization procedure. Our findings compared to currently published methods demonstrate that continuous complex real-time measurements and analyses are required to produce an optimal scaffold for cardiac regeneration.

Sequential multimodal microscopic imaging and biaxial mechanical testing of living multicomponent tissue constructs.

Understanding relationships between mechanical stimuli and cellular responses require measurements of evolving tissue structure and mechanical properties. We developed a 3D tissue bioreactor that couples to both the stage of a custom multimodal microscopy system and a biaxial mechanical testing platform. Time dependent changes in microstructure and mechanical properties of fibroblast seeded cruciform fibrin gels were investigated while cultured under either anchored (1.0:1.0 stretch ratio) or strip biaxial (1.0:1.1) conditions. A multimodal nonlinear optical microscopy-optical coherence microscopy (NLOM-OCM) system was used to delineate noninvasively the relative spatial distributions of original fibrin, deposited collagen, and fibroblasts during month long culture. Serial in-culture mechanical testing was also performed to track the evolution of bulk mechanical properties under sterile conditions. Over the month long time course, seeded cells and deposited collagen were randomly distributed in equibiaxially anchored constructs, but exhibited preferential alignment parallel to the direction of the 10% stretch in constructs cultured under strip biaxial stretch. Surprisingly, both anchored and strip biaxial stretched constructs exhibited isotropic mechanical properties (including progressively increasing stiffness) despite developing a very different collagen microstructural organization. In summary, our biaxial bioreactor system integrating both NLOM-OCM and mechanical testing provided complementary information on microstructural organization and mechanical properties and, thus, may enable greater fundamental understanding of relationships between engineered soft tissue mechanics and mechanobiology.

Dynamic multicomponent engineered tissue reorganization and matrix deposition measured with an integrated nonlinear optical microscopy-optical coherence microscopy system.

Multicomponent tissue models are viable tools to better understand cell responses in complex environments, but present challenges when investigated with live cell microscopy noninvasively. In this study, integrated nonlinear optical microscopy-optical coherence microscopy (NLOM-OCM) was used to characterize cell interactions within three-dimensional (3-D), multicomponent extracellular matrices. In fibrin-collagen mixtures, 3T3 fibroblasts were observed to recruit both fibrin and collagen fibers while remodeling matrices. Also, NLOM-OCM was used to observe collagen deposition by neonatal human dermal fibroblasts within originally fibrin matrices over an extended time. It was observed that preferentially aligned collagen deposition could be achieved with aligned fibroblasts but that cell alignment could be achieved without aligning the extant extracellular matrix. In summary, this multimodel imaging system has potential for both real-time and longitudinal imaging of living 3-D cultures, which is particularly important for evaluating cell microenvironments in composite scaffolds or serial characterization of engineered tissue constructs during culture.

An ensemble rank learning approach for gene prioritization.

Several different computational approaches have been developed to solve the gene prioritization problem. We intend to use the ensemble boosting learning techniques to combine variant computational approaches for gene prioritization in order to improve the overall performance. In particular we add a heuristic weighting function to the Rankboost algorithm according to: 1) the absolute ranks generated by the adopted methods for a certain gene, and 2) the ranking relationship between all gene-pairs from each prioritization result. We select 13 known prostate cancer genes in OMIM database as training set and protein coding gene data in HGNC database as test set. We adopt the leave-one-out strategy for the ensemble rank boosting learning. The experimental results show that our ensemble learning approach outperforms the four gene-prioritization methods in ToppGene suite in the ranking results of the 13 known genes in terms of mean average precision, ROC and AUC measures.

In vivo measurement of proximal pulmonary artery elastic modulus in the neonatal calf model of pulmonary hypertension: development and ex vivo validation.

Developing clinical work suggests that vascular stiffening plays a role in the progression of pulmonary hypertension (PH), while recent studies in animal models of hypoxic PH have found significant proximal vascular stiffening in the diseased population. Here, we develop and validate a minimally invasive, clinically realizable method to estimate the local elastic modulus of the proximal pulmonary arteries from pressure-diameter (PD) data. PD measurements were made in the main pulmonary arteries of 16 calves; lumen diameter was assessed using color M-mode tissue Doppler imaging ultrasound, while pressure was measured via catheter. Two methods corresponding to thin-walled pressure vessel theory ("thin") and Lame's equation for a thick-walled cylinder ("thick") were used to approximate the artery elastic modulus from PD measurements. The harvested arteries were tested independently to determine their "true" ex vivo elastic modulus and stiffness. Both approximations displayed excellent correlation with ex vivo elastic modulus of the calf main pulmonary artery (thin r(2) = 0.811; thick r(2) = 0.844; both P < 0.01). Bland-Altman analysis indicated that the thick-walled approximation has better overall agreement with ex vivo modulus. The approximations displayed quantitatively distinct regression slopes that were statistically different (P = 0.02). The elastic modulus of the main pulmonary artery can be reasonably estimated from combined color M-mode tissue Doppler imaging ultrasound and catheter pressure measurements in calves. Such measurements may be a valuable tool in the diagnosis and treatment of human PH.

ULTRASHORT PULSE MULTISPECTRAL NONLINEAR OPTICAL MICROSCOPY.

Ultrashort pulse, multispectral nonlinear optical microscopy (NLOM) is developed and used to image, simultaneously, a mixed population of cells expressing different fluorescent protein mutants in a 3D tissue model of angiogenesis. Broadband, sub-10-fs pulses are used to excite multiple fluorescent proteins and generate second harmonic in collagen simultaneously. A 16-channel multispectral detector is used to delineate the multiple nonlinear optical signals, pixel by pixel, in NLOM. The ability to image multiple fluorescent protein mutants and collagen, simultaneously, enables serial measurements of cell-cell and cell-matrix interactions in our 3D tissue model and characterization of fundamental processes in angiogenic morphogenesis.

Nonlinear optical microscopy reveals invading endothelial cells anisotropically alter three-dimensional collagen matrices.

The interactions between endothelial cells (ECs) and the extracellular matrix (ECM) are fundamental in mediating various steps of angiogenesis, including cell adhesion, migration and sprout formation. Here, we used a noninvasive and non-destructive nonlinear optical microscopy (NLOM) technique to optically image endothelial sprouting morphogenesis in three-dimensional (3D) collagen matrices. We simultaneously captured signals from collagen fibers and endothelial cells using second harmonic generation (SHG) and two-photon excited fluorescence (TPF), respectively. Dynamic 3D imaging revealed EC interactions with collagen fibers along with quantifiable alterations in collagen matrix density elicited by EC movement through and morphogenesis within the matrix. Specifically, we observed increased collagen density in the area between bifurcation points of sprouting structures and anisotropic increases in collagen density around the perimeter of lumenal structures, but not advancing sprout tips. Proteinase inhibition studies revealed membrane-associated matrix metalloproteinase were utilized for sprout advancement and lumen expansion. Rho-associated kinase (p160ROCK) inhibition demonstrated that the generation of cell tension increased collagen matrix alterations. This study followed sprouting ECs within a 3D matrix and revealed that the advancing structures recognize and significantly alter their extracellular environment at the periphery of lumens as they progress.

Pulmonary vascular input impedance is a combined measure of pulmonary vascular resistance and stiffness and predicts clinical outcomes better than pulmonary vascular resistance alone in pediatric patients with pulmonary hypertension.

BACKGROUND: Pulmonary vascular resistance (PVR) is the current standard for evaluating reactivity in children with pulmonary arterial hypertension (PAH). However, PVR measures only the mean component of right ventricular afterload and neglects pulsatile effects. We recently developed and validated a method to measure pulmonary vascular input impedance, which revealed excellent correlation between the zero harmonic impedance value and PVR and suggested a correlation between higher-harmonic impedance values and pulmonary vascular stiffness. Here we show that input impedance can be measured routinely and easily in the catheterization laboratory, that impedance provides PVR and pulmonary vascular stiffness from a single measurement, and that impedance is a better predictor of disease outcomes compared with PVR. METHODS: Pressure and velocity waveforms within the main pulmonary artery were measured during right heart catheterization of patients with normal pulmonary artery hemodynamics (n = 14) and those with PAH undergoing reactivity evaluation (49 subjects, 95 conditions). A correction factor needed to transform velocity into flow was obtained by calibrating against cardiac output. Input impedance was obtained off-line by dividing Fourier-transformed pressure and flow waveforms. RESULTS: Exceptional correlation was found between the indexed zero harmonic of impedance and indexed PVR (y = 1.095x + 1.381, R2 = 0.9620). In addition, the modulus sum of the first 2 harmonics of impedance was found to best correlate with indexed pulse pressure over stroke volume (y = 13.39x - 0.8058, R2 = 0.7962). Among a subset of patients with PAH (n = 25), cumulative logistic regression between outcomes to total indexed impedance was better (R(L)2 = 0.4012) than between outcomes and indexed PVR (R(L)2 = 0.3131). CONCLUSIONS: Input impedance can be consistently and easily obtained from pulse-wave Doppler and a single catheter pressure measurement, provides comprehensive characterization of the main components of RV afterload, and better predicts patient outcomes compared with PVR alone.

Noninvasive Doppler tissue measurement of pulmonary artery compliance in children with pulmonary hypertension.

BACKGROUND: We have shown previously that input impedance of the pulmonary vasculature provides a comprehensive characterization of right ventricular afterload by including compliance. However, impedance-based compliance assessment requires invasive measurements. Here, we develop and validate a noninvasive method to measure pulmonary artery (PA) compliance using ultrasound color M-mode (CMM) Doppler tissue imaging (DTI). METHODS: Dynamic compliance (C(dyn)) of the PA was obtained from CMM DTI and continuous wave Doppler measurement of the tricuspid regurgitant velocity. C(dyn) was calculated as: [(D(s) - D(d))/(D(d) x P(s))] x 10(4); where D(s) = systolic diameter, D(d) = diastolic diameter, and P(s) = systolic pressure. The method was validated both in vitro and in 13 patients in the catheterization laboratory, and then tested on 27 pediatric patients with pulmonary hypertension, with comparison with 10 age-matched control subjects. C(dyn) was also measured in an additional 13 patients undergoing reactivity studies. RESULTS: Instantaneous diameter measured using CMM DTI agreed well with intravascular ultrasound measurements in the in vitro models. Clinically, C(dyn) calculated by CMM DTI agreed with C(dyn) calculated using invasive techniques (23.4 +/- 16.8 vs 29.1 +/- 20.6%/100 mm Hg; P = not significant). Patients with pulmonary hypertension had significantly lower peak wall velocity values and lower C(dyn) values than control subjects (P < .01). C(dyn) values followed an exponentially decaying relationship with PA pressure, indicating the nonlinear stress-strain behavior of these arteries. Reactivity in C(dyn) agreed with reactivity measured using impedance techniques. CONCLUSION: The C(dyn) method provides a noninvasive means of assessing PA compliance and should be useful as an additional measure of vascular reactivity subsequent to pulmonary vascular resistance in patients with pulmonary hypertension.