Xuejun Wen

GDNF modifies reactive astrogliosis allowing robust axonal regeneration through Schwann cell-seeded guidance channels after spinal cord injury

Reactive astrogliosis impedes axonal regeneration after injuries to the mammalian central nervous system (CNS). Here we report that glial cell line-derived neurotrophic factor (GDNF), combined with transplanted Schwann cells (SCs), effectively reversed the inhibitory properties of astrocytes at graft-host interfaces allowing robust axonal regeneration, concomitant with vigorous migration of host astrocytes into SC-seeded semi-permeable guidance channels implanted into a right-sided spinal cord hemisection at the 10th thoracic (T10) level. Within the graft, migrated host astrocytes were in close association with regenerated axons. Astrocyte processes extended parallel to the axons, implying that the migrated astrocytes were not inhibitory and might have promoted directional growth of regenerated axons. In vitro, GDNF induced migration of SCs and astrocytes toward each other in an astrocyte-SC confrontation assay. GDNF also enhanced migration of astrocytes on a SC monolayer in an inverted coverslip migration assay, suggesting that this effect is mediated by direct cell-cell contact between the two cell types. Morphologically, GDNF administration reduced astrocyte hypertrophy and induced elongated process extension of these cells, similar to what was observed in vivo. Notably, GDNF treatment significantly reduced production of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs), two hallmarks of astrogliosis, in both the in vivo and in vitro models. Thus, our study demonstrates a novel role of GDNF in modifying spinal cord injury (SCI)-induced astrogliosis resulting in robust axonal regeneration in adult rats.

Helium ion microscopy for high-resolution visualization of the articular cartilage collagen network.

The articular cartilage collagen network is an important research focus because network disruption results in cartilage degeneration and patient disability. The recently introduced helium ion microscope (HIM), with its smaller probe size, longer depth of field and charge neutralization, has the potential to overcome the inherent limitations of electron microscopy for visualization of collagen network features, particularly at the nanoscale. In this study, we evaluated the capabilities of the helium ion microscope for high‐resolution visualization of the articular cartilage collagen network. Images of rabbit knee cartilage were acquired with a helium ion microscope; comparison images were acquired with a field emission scanning electron microscope (FE‐SEM) and a transmission electron microscope (TEM). Sharpness of example high‐resolution helium ion microscope and field emission scanning electron microscope images was quantified using the 25–75% rise distance metric. The helium ion microscope was able to acquire high‐resolution images with unprecedented clarity, with greater sharpness and three‐dimensional‐like detail of nanoscale fibril morphologies and fibril connections, in samples without conductive coatings. These nanoscale features could not be resolved by field emission scanning electron microscopy, and three‐dimensional network structure could not be visualized with transmission electron microscopy. The nanoscale three‐dimensional‐like visualization capabilities of the helium ion microscope will enable new avenues of investigation in cartilage collagen network research.

An In Vitro Model of the Glomerular Capillary Wall Using Electrospun Collagen Nanofibres in a Bioartificial Composite Basement Membrane

The filtering unit of the kidney, the glomerulus, contains capillaries whose walls function as a biological sieve, the glomerular filtration barrier. This comprises layers of two specialised cells, glomerular endothelial cells (GEnC) and podocytes, separated by a basement membrane. Glomerular filtration barrier function, and dysfunction in disease, remains incompletely understood, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised human GEnC and podocytes. However, because the glomerular filtration barrier functions as a whole, it is necessary to develop three dimensional co-culture models to maximise the benefit of the availability of these cells. Here we have developed the first two tri-layer models of the glomerular capillary wall. The first is based on tissue culture inserts and provides evidence of cell-cell interaction via soluble mediators. In the second model the synthetic support of the tissue culture insert is replaced with a novel composite bioartificial membrane. This consists of a nanofibre membrane containing collagen I, electrospun directly onto a micro-photoelectroformed fine nickel supporting mesh. GEnC and podocytes grew in monolayers on either side of the insert support or the novel membrane to form a tri-layer model recapitulating the human glomerular capillary in vitro. These models will advance the study of both the physiology of normal glomerular filtration and of its disruption in glomerular disease.

Bioengineering Strategies for Designing Targeted Cancer Therapies

The goals of bioengineering strategies for targeted cancer therapies are (1) to deliver a high dose of an anticancer drug directly to a cancer tumor, (2) to enhance drug uptake by malignant cells, and (3) to minimize drug uptake by nonmalignant cells. Effective cancer-targeting therapies will require both passive- and active-targeting strategies and a thorough understanding of physiologic barriers to targeted drug delivery. Designing a targeted therapy includes the selection and optimization of a nanoparticle delivery vehicle for passive accumulation in tumors, a targeting moiety for active receptor-mediated uptake, and stimuli-responsive polymers for control of drug release. The future direction of cancer targeting is a combinatorial approach, in which targeting therapies are designed to use multiple-targeting strategies. The combinatorial approach will enable combination therapy for delivery of multiple drugs and dual ligand targeting to improve targeting specificity. Targeted cancer treatments in development and the new combinatorial approaches show promise for improving targeted anticancer drug delivery and improving treatment outcomes.

Formation of Well-defined Embryoid Bodies from Dissociated Human Induced Pluripotent Stem Cells using Microfabricated Cell-repellent Microwell Arrays

A simple, scalable, and reproducible technology that allows direct formation of large numbers of homogeneous and synchronized embryoid bodies (EBs) of defined sizes from dissociated human induced pluripotent stem cells (hiPSCs) was developed. Non-cell-adhesive hydrogels were used to create round-bottom microwells to host dissociated hiPSCs. No Rho-associated kinase inhibitor (ROCK-i), or centrifugation was needed and the side effects of ROCK-i can be avoided. The key requirement for the successful EB formation in addition to the non-cell-adhesive round-bottom microwells is the input cell density per microwell. Too few or too many cells loaded into the microwells will compromise the EB formation process. In parallel, we have tested our microwell-based system for homogeneous hEB formation from dissociated human embryonic stem cells (hESCs). Successful production of homogeneous hEBs from dissociated hESCs in the absence of ROCK-i and centrifugation was achieved within an optimal range of input cell density per microwell. Both the hiPSC- and hESC-derived hEBs expressed key proteins characteristic of all the three developmental germ layers, confirming their EB identity. This novel EB production technology may represent a versatile platform for the production of homogeneous EBs from dissociated human pluripotent stem cells (hPSCs).

Highly Aligned Polymer Nanofiber Structures: Fabrication and Applications in Tissue Engineering

Many types of tissue in the body, such as nerve, muscle, tendon, ligament, bone, and blood vessels, rely on a highly organized microstructure in order to impart their desired functionality. Cell and extracellular matrix (ECM) alignment in these tissues allows for increased mechanical strength and cell communication. In tissue engineering, aligned polymer nanofibers can be used to take on the role of natural ECM fibers in order to provide mechanical strength, sites for cell attachment, and modulation of cell behavior via morphological cues. A wide variety of physical and electrostatic techniques are available for assembly of aligned nanofiber structures, and many of these structures have been evaluated as tissue engineering scaffolds. It is widely understood that aligned microstructure induces an aligned morphology in most cell types, but aligned nanofibrous topography also influences other cell behaviors such as differentiation, gene expression, and ECM deposition. With a greater understanding of aligned nanofiber scaffold fabrication techniques, and cell interactions with these scaffolds, researchers may be able to overcome current challenges and develop better strategies for regenerating aligned tissues.

Fabrication of a biomimetic elastic intervertebral disk scaffold using additive manufacturing

A custom-designed three-dimensional additive manufacturing device was developed to fabricate scaffolds for intervertebral disk (IVD) regeneration. This technique integrated a computer with a device capable of 3D movement allowing for precise motion and control over the polymer scaffold resolution. IVD scaffold structures were designed using computer-aided design to resemble the natural IVD structure. Degradable polyurethane (PU) was used as an elastic scaffold construct to mimic the elastic nature of the native IVD tissue and was deposited at a controlled rate using ultra-fine micropipettes connected to a syringe pump. The elastic PU was extruded directly onto a collecting substrate placed on a freezing stage. The three-dimensional movement of the computer-controlled device combined with the freezing stage enabled precise control of polymer deposition using extrusion. The addition of the freezing stage increased the polymer solution viscosity and hardened the polymer solution as it was extruded out of the micropipette tip. This technique created scaffolds with excellent control over macro- and micro-structure to influence cell behavior, specifically for cell adhesion, proliferation, and alignment. Concentric lamellae were printed at a high resolution to mimic the native shape and structure of the IVD. Seeded cells aligned along the concentric lamellae and acquired cell morphology similar to native tissue in the outer portion of the IVD. The fabricated scaffolds exhibited elastic behavior during compressive and shear testing, proving that the scaffolds could support loads with proper fatigue resistance without permanent deformation. Additionally, the mechanical properties of the scaffolds were comparable to those of native IVD tissue.

Manipulating neural-stem-cell mobilization and migration in vitro.

Neural stem-cell transplantation is a promising strategy for the treatment of neural diseases and injuries, since the central nervous system (CNS) has a very limited capacity to repopulate the lost cells. Transplantation strategies face many difficulties including low viability, lack of control of stem-cell fate, and low levels of cell engraftment after transplantation. An alternative strategy for CNS repair without transplantation is using endogenous neural stem cells (NSCs) and precursor cells. Hepatocyte growth factor (HGF), a pleiotropic cytokine of mesenchymal origin, exerts a strong chemoattractive effect on stem cells. Leukemia inhibitory factor (LIF), a key regulator for stem-cell proliferation, mobilization, and fate choices, is currently being characterized for endogenous NSC manipulation for brain regeneration. In this study, HGF and LIF have been loaded into hydrogels and degradable nanoparticles, respectively, for sustained, long-term, localized delivery. We examine the use of HGF-loaded hydrogels and LIF-loaded nanoparticles for manipulating migration and mobilization of human NSCs in vitro. The combination of LIF-loaded nanoparticles and HGF-loaded hydrogels significantly mobilized hNSCs and promoted their migration in vitro. Studies are in progress to evaluate endogenous NSC mobilization and migration in vivo with simultaneous, controlled delivery of LIF at the natural reservoir of endogenous NSCs and HGF at the injury or disease site for in situ tissue regeneration.

A Novel Method to Precisely Assemble Loose Nanofiber Structures for Regenerative Medicine Applications

Polymer nanofibers are favorable for tissue engineering scaffolds because of their high surface-to-volume ratio and biomimicry of the extracellular matrix. Random and uniaxially oriented polymer nanofibers are easily fabricated by conventional electrospinning techniques; however, control over fiber organization within nanofiber structures is limited when they are collected directly from an electrospinning jet. The regenerative medicine applications of electrospun scaffolds could be expanded by developing assembly methods that allow better control of fiber organization. Here, a novel technique is presented that utilizes parallel automated tracks to orient and collect nanofibers from an electrospinning jet. The stabilized fibers are then subsequently assembled into desirable structures. It is difficult to assemble complex structures directly from an electrospinning jet because of high electrical charge and velocities, so this technology adds an intermediate step where nanofibers are immobilized on automated tracks. The result is a continuous steady-state delivery of static stabilized nanofibers that provides a unique and promising platform for automated post processing into useful nanofiber structures. This technique also allows for an indefinite amount of time, as determined by design parameters, for fibers to dry or cool before they contact other nanofibers in the collection site, thus eliminating potential for fiber-to-fiber adhesions even with slow evaporating solvents or high-temperature melts. To demonstrate potential in regenerative medicine applications, several nanofiber structures were fabricated, including: 2D structures with well-controlled fiber density; 3D loosely assembled aligned nanofiber structures with good cell penetration properties; and, complex layer-by-layer 3D aligned fiber structures assembled by integration with post-processing techniques.

Scalable Differentiation of Human iPSCs in a Multicellular Spheroid-based 3D Culture into Hepatocyte-like Cells through Direct Wnt/β-catenin Pathway Inhibition

Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields, cellular heterogeneity, and limited scalability. In the present study, we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes, secreted hepatic proteins such as Albumin, Alpha Fetoprotein, and Fibrinogen, metabolized ammonia, and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes, such as LDL storage and uptake, ICG uptake and release, and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure, providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1.