Karina H. Nakayama

Tissue Specificity of Decellularized Rhesus Monkey Kidney and Lung Scaffolds

Initial steps in establishing an optimal strategy for functional bioengineered tissues is generation of three-dimensional constructs containing cells with the appropriate organization and phenotype. To effectively utilize rhesus monkey decellularized kidney scaffolds, these studies evaluated two key parameters: (1) residual scaffold components after decellularization including proteomics analysis, and (2) the use of undifferentiated human embryonic stem cells (hESCs) for recellularization in order to explore cellular differentiation in a tissue-specific manner. Sections of kidney and lung were selected for a comparative evaluation because of their similar pattern of organogenesis. Proteomics analysis revealed the presence of growth factors and antimicrobial proteins as well as stress proteins and complement components. Immunohistochemistry of recellularized kidney scaffolds showed the generation of Cytokeratin+ epithelial tubule phenotypes throughout the scaffold that demonstrated a statistically significant increase in expression of kidney-associated genes compared to baseline hESC gene expression. Recellularization of lung scaffolds showed that cells lined the alveolar spaces and demonstrated statistically significant upregulation of key lung-associated genes. However, overall expression of kidney and lung-associated markers was not statistically different when the kidney and lung recellularized scaffolds were compared. These results suggest that decellularized scaffolds have an intrinsic spatial ability to influence hESC differentiation by physically shaping cells into tissue-appropriate structures and phenotypes, and that additional approaches may be needed to ensure consistent recellularization throughout the matrix.

Near-Infrared II Fluorescence for Imaging Hindlimb Vessel Regeneration With Dynamic Tissue Perfusion Measurement

Background—Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here, we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000–1400 nm) of photon wavelengths. Methods and Results—Because of the reduced photon scattering of NIR-II fluorescence compared with traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microscopic computed tomography. Furthermore, imaging during 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (P<0.005), which was confirmed by histological analysis of microvascular density. Moreover, the tissue perfusion in the ischemic hindlimb could be quantitatively measured by the dynamic NIR-II method, revealing the temporal kinetics of blood flow recovery that resembled microbead-based blood flowmetry and laser Doppler blood spectroscopy. Conclusions—The penetration depth of millimeters, high spatial resolution, and fast acquisition rate of NIR-II imaging make it a useful imaging tool for murine models of vascular disease.

Role of Extracellular Matrix Signaling Cues in Modulating Cell Fate Commitment for Cardiovascular Tissue Engineering

It is generally agreed that engineered cardiovascular tissues require cellular interactions with the local milieu. Within the microenvironment, the extracellular matrix (ECM) is an important support structure that provides dynamic signaling cues in part through its chemical, physical, and mechanical properties. In response to ECM factors, cells activate biochemical and mechanotransduction pathways that modulate their survival, growth, migration, differentiation, and function. This Review describes the role of ECM chemical composition, spatial patterning, and mechanical stimulation in the specification of cardiovascular lineages, with a focus on stem cell differentiation, direct transdifferentiation, and endothelial-to-mesenchymal transition. The translational application of ECMs is discussed in the context of cardiovascular tissue engineering and regenerative medicine.

Aligned-Braided Nanofibrillar Scaffold with Endothelial Cells Enhances Arteriogenesis

The objective of this study was to enhance the angiogenic capacity of endothelial cells (ECs) using nanoscale signaling cues from aligned nanofibrillar scaffolds in the setting of tissue ischemia. Thread-like nanofibrillar scaffolds with porous structure were fabricated from aligned-braided membranes generated under shear from liquid crystal collagen solution. Human ECs showed greater outgrowth from aligned scaffolds than from nonpatterned scaffolds. Integrin α1 was in part responsible for the enhanced cellular outgrowth on aligned nanofibrillar scaffolds, as the effect was abrogated by integrin α1 inhibition. To test the efficacy of EC-seeded aligned nanofibrillar scaffolds in improving neovascularization in vivo, the ischemic limbs of mice were treated with EC-seeded aligned nanofibrillar scaffold; EC-seeded nonpatterned scaffold; ECs in saline; aligned nanofibrillar scaffold alone; or no treatment. After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion recovery when treated with EC-seeded aligned nanofibrillar scaffolds, in comparison to ECs in saline or no treatment. In ischemic hindlimbs treated with scaffolds seeded with human ECs derived from induced pluripotent stem cells (iPSC-ECs), single-walled carbon nanotube (SWNT) fluorophores were systemically delivered to quantify microvascular density after 28 days. Near infrared-II (NIR-II, 1000-1700 nm) imaging of SWNT fluorophores demonstrated that iPSC-EC-seeded aligned scaffolds group showed significantly higher microvascular density than the saline or cells groups. These data suggest that treatment with EC-seeded aligned nanofibrillar scaffolds improved blood perfusion and arteriogenesis, when compared to treatment with cells alone or scaffold alone, and have important implications in the design of therapeutic cell delivery strategies.

Bilayered vascular graft derived from human induced pluripotent stem cells with biomimetic structure and function

BACKGROUND We developed an aligned bi-layered vascular graft derived from human induced pluripotent stem cells (iPSCs) that recapitulates the cellular composition, orientation, and anti-inflammatory function of blood vessels. MATERIALS & METHODS The luminal layer consisted of longitudinal-aligned nanofibrillar collagen containing primary endothelial cells (ECs) or iPSC-derived ECs (iPSC-ECs). The outer layer contained circumferentially oriented nanofibrillar collagen with primary smooth muscle cells (SMCs) or iPSC-derived SMCs(iPSC-SMCs). RESULTS On the aligned scaffolds, cells organized F-actin assembly within 8º from the direction of nanofibrils. When compared to randomly-oriented scaffolds, EC-seeded aligned scaffolds had significant reduced inflammatory response, based on adhesivity to monocytes. CONCLUSION This study highlights the importance of anisotropic scaffolds in directing cell form and function, and has therapeutic significance as physiologically relevant blood vessels.

Nanoscale Patterning of Extracellular Matrix Alters Endothelial Function under Shear Stress

The role of nanotopographical extracellular matrix (ECM) cues in vascular endothelial cell (EC) organization and function is not well-understood, despite the composition of nano- to microscale fibrillar ECMs within blood vessels. Instead, the predominant modulator of EC organization and function is traditionally thought to be hemodynamic shear stress, in which uniform shear stress induces parallel-alignment of ECs with anti-inflammatory function, whereas disturbed flow induces a disorganized configuration with pro-inflammatory function. Since shear stress acts on ECs by applying a mechanical force concomitant with inducing spatial patterning of the cells, we sought to decouple the effects of shear stress using parallel-aligned nanofibrillar collagen films that induce parallel EC alignment prior to stimulation with disturbed flow resulting from spatial wall shear stress gradients. Using real time live-cell imaging, we tracked the alignment, migration trajectories, proliferation, and anti-inflammatory behavior of ECs when they were cultured on parallel-aligned or randomly oriented nanofibrillar films. Intriguingly, ECs cultured on aligned nanofibrillar films remained well-aligned and migrated predominantly along the direction of aligned nanofibrils, despite exposure to shear stress orthogonal to the direction of the aligned nanofibrils. Furthermore, in stark contrast to ECs cultured on randomly oriented films, ECs on aligned nanofibrillar films exposed to disturbed flow had significantly reduced inflammation and proliferation, while maintaining intact intercellular junctions. This work reveals fundamental insights into the importance of nanoscale ECM interactions in the maintenance of endothelial function. Importantly, it provides new insight into how ECs respond to opposing cues derived from nanotopography and mechanical shear force and has strong implications in the design of polymeric conduits and bioengineered tissues.

Targeted delivery of human iPS-ECs overexpressing IL-8 receptors inhibits neointimal and inflammatory responses to vascular injury in the rat

Interleukin-8 (IL8) is highly expressed by injured arteries in a variety of diseases and is a chemoattractant for neutrophils which express IL8 receptors IL8RA and RB (IL8RA/B) on their membranes. Neutrophils interact with the damaged endothelium and initiate an inflammatory cascade at the site of injury. We have generated a novel translational targeted cell therapy for acute vascular injury using adenoviral vectors to overexpress IL8RA/B and green fluorescent protein (GFP) on the surface of endothelial cells (ECs) derived from human induced pluripotent stem cells (HiPS-IL8RA/B-ECs). We hypothesize that HiPS-IL8RA/B-ECs transfused intravenously into rats with balloon injury of the carotid artery will target to the injured site and compete with neutrophils, thus inhibiting inflammation and neointima formation. Young adult male Sprague-Dawley rats underwent balloon injury of the right carotid artery and received intravenous transfusion of saline vehicle, 1.5 × 10(6) HiPS-ECs, 1.5 × 10(6) HiPS-Null-ECs, or 1.5 × 10(6) HiPS-IL8RA/B-ECs immediately after endoluminal injury. Tissue distribution of HiPS-IL8RA/B-ECs was analyzed by a novel GFP DNA qPCR method. Cytokine and chemokine expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 h postinjury by ELISA and immunohistochemistry, respectively. Neointimal, medial areas, and reendothelialization were measured 14 days postinjury. HiPS-IL8RA/B-ECs homed to injured arteries, inhibited inflammatory mediator expression and inflammatory cell infiltration, accelerated reendothelialization, and attenuated neointima formation after endoluminal injury while control HiPS-ECs and HiPS-Null-ECs did not. HiPS-IL8RA/B-ECs transfused into rats with endoluminal carotid artery injury target to the injured artery and provide a novel strategy to treat vascular injury.

Use of Large Animal and Nonhuman Primate Models for Cell Therapy and Tissue Engineering

The intent of this chapter is to highlight the contributions of large animal models including pigs, sheep, goats, dogs, and nonhuman primates. These species are crucial for clinical translation of new cell and tissue engineering approaches for the treatment of human diseases. Choice of species, age, and model validation are necessary to ensure outcomes are predictive, and recapitulate human development, anatomy, physiology, and disease. The overriding objective is to select a model that can reliably assess the safety and efficacy of new therapies beyond the discovery phase and to obtain results that can be translated to clinical trials in children and adults.

Rehabilitative exercise and spatially patterned nanofibrillar scaffolds enhance vascularization and innervation following volumetric muscle loss

Muscle regeneration can be permanently impaired by traumatic injuries, despite the high regenerative capacity of skeletal muscle. Implantation of engineered biomimetic scaffolds to the site of muscle ablation may serve as an attractive off-the-shelf therapeutic approach. The objective of the study was to histologically assess the therapeutic benefit of a three-dimensional spatially patterned collagen scaffold, in conjunction with rehabilitative exercise, for treatment of volumetric muscle loss. To mimic the physiologic organization of skeletal muscle, which is generally composed of myofibers aligned in parallel, three-dimensional parallel-aligned nanofibrillar collagen scaffolds were fabricated. When implanted into the ablated murine tibialis anterior muscle, the aligned nanofibrillar scaffolds, in conjunction with voluntary caged wheel exercise, significantly improved the density of perfused microvessels, in comparison to treatments of the randomly oriented nanofibrillar scaffold, decellularized scaffold, or in the untreated control group. The abundance of neuromuscular junctions was 19-fold higher when treated with aligned nanofibrillar scaffolds in conjunction with exercise, in comparison to treatment of aligned scaffold without exercise. Although, the density of de novo myofibers was not significantly improved by aligned scaffolds, regardless of exercise activity, the cross-sectional area of regenerating myofibers was increased by > 60% when treated with either aligned and randomly oriented scaffolds, in comparison to treatment of decellularized scaffold or untreated controls. These findings demonstrate that voluntary exercise improved the regenerative effect of aligned scaffolds by augmenting neurovascularization, and have important implications in the design of engineered biomimetic scaffolds for treatment of traumatic muscle injury.Muscle injury: exercise boosts regenerative potential of engineered collagen scaffoldsA collagen scaffold designed to mimic skeletal muscle, together with rehabilitative exercise, can help regenerate nerves and blood vessels following traumatic muscle injury. Ngan Huang from Stanford University, California, USA, and colleagues created scaffolding composed of collagen nanofibers aligned in parallel, as natural muscle fibers are. They implanted these specially patterned collagen constructs into the shins of mice that had no tibialis anterior muscles. Mice given the opportunity to exercise formed far more nerve connections in their injured muscles compared to mice without exercise wheels in their cages. Active mice also developed significantly more blood vessels in their injured muscles with the parallel-aligned scaffolds compared to other animals with randomly oriented scaffolds, decellularized scaffolds or no implant at all. The findings highlight the potential of combining exercise and biomimetic scaffolds to treat muscle trauma.