A therapeutic convection-enhanced macroencapsulation device for enhancing ß cell viability and insulin secretion.

Islet transplantation for type 1 diabetes treatment has been limited by the need for lifelong immunosuppression regimens. This challenge has prompted the development of macroencapsulation devices (MEDs) to immunoprotect the transplanted islets. While promising, conventional MEDs are faced with insufficient transport of oxygen, glucose, and insulin because of the reliance on passive diffusion. Hence, these devices are constrained to two-dimensional, wafer-like geometries with limited loading capacity to maintain cells within a distance of passive diffusion. We hypothesized that convective nutrient transport could extend the loading capacity while also promoting cell viability, rapid glucose equilibration, and the physiological levels of insulin secretion. Here, we showed that convective transport improves nutrient delivery throughout the device and affords a three-dimensional capsule geometry that encapsulates 9.7-fold-more cells than conventional MEDs. Transplantation of a convection-enhanced MED (ceMED) containing insulin-secreting ß cells into immunocompetent, hyperglycemic rats demonstrated a rapid, vascular-independent, and glucose-stimulated insulin response, resulting in early amelioration of hyperglycemia, improved glucose tolerance, and reduced fibrosis. Finally, to address potential translational barriers, we outlined future steps necessary to optimize the ceMED design for long-term efficacy and clinical utility.

Magnetic Control of Axon Navigation in Reprogrammed Neurons.

While neural cell transplantation represents a promising therapy for neurodegenerative diseases, the formation of functional networks of transplanted cells with host neurons constitutes one of the challenging steps. Here, we introduce a magnetic guidance methodology that controls neurite growth signaling via magnetic nanoparticles (MNPs) conjugated with antibodies targeting the deleted in colorectal cancer (DCC) receptor (DCC-MNPs). Activation of the DCC receptors by clusterization and subsequent axonal growth of the induced neuronal (iN) cells was performed in a spatially controlled manner. In addition to the directionality of the magnetically controlled axon projection, axonal growth of the iN cells assisted the formation of functional connections with pre-existing primary neurons. Our results suggest magnetic guidance as a strategy for improving neuronal connectivity by spatially guiding the axonal projections of transplanted neural cells for synaptic interactions with the host tissue.

Three-Dimensional Electroconductive Hyaluronic Acid Hydrogels Incorporated with Carbon Nanotubes and Polypyrrole by Catechol-Mediated Dispersion Enhance Neurogenesis of Human Neural Stem Cells.

Electrically conductive hyaluronic acid (HA) hydrogels incorporated with single-walled carbon nanotubes (CNTs) and/or polypyrrole (PPy) were developed to promote differentiation of human neural stem/progenitor cells (hNSPCs). The CNT and PPy nanocomposites, which do not easily disperse in aqueous phases, dispersed well and were efficiently incorporated into catechol-functionalized HA (HA-CA) hydrogels by the oxidative catechol chemistry used for hydrogel cross-linking. The prepared electroconductive HA hydrogels provided dynamic, electrically conductive three-dimensional (3D) extracellular matrix environments that were biocompatible with hNSPCs. The HA-CA hydrogels containing CNT and/or PPy significantly promoted neuronal differentiation of human fetal neural stem cells (hfNSCs) and human induced pluripotent stem cell-derived neural progenitor cells (hiPSC-NPCs) with improved electrophysiological functionality when compared to differentiation of these cells in a bare HA-CA hydrogel without electroconductive motifs. Calcium channel expression was upregulated, depolarization was activated, and intracellular calcium influx was increased in hNSPCs that were differentiated in 3D electroconductive HA-CA hydrogels; these data suggest a potential mechanism for stem cell neurogenesis. Overall, our bioinspired, electroconductive HA hydrogels provide a promising cell-culture platform and tissue-engineering scaffold to improve neuronal regeneration.

Mussel Adhesion-Inspired Reverse Transfection Platform Enhances Osteogenic Differentiation and Bone Formation of Human Adipose-Derived Stem Cells.

Using small interfering RNA (siRNA) to regulate gene expression is an emerging strategy for stem cell manipulation to improve stem cell therapy. However, conventional methods of siRNA delivery into stem cells based on solution-mediated transfection are limited due to low transfection efficiency and insufficient duration of cell-siRNA contact during lengthy culturing protocols. To overcome these limitations, a bio-inspired polymer-mediated reverse transfection system is developed consisting of implantable poly(lactic-co-glycolic acid) (PLGA) scaffolds functionalized with siRNA-lipidoid nanoparticle (sLNP) complexes via polydopamine (pDA) coating. Immobilized sLNP complexes are stably maintained without any loss of siRNA on the pDA-coated scaffolds for 2 weeks, likely due to the formation of strong covalent bonds between amine groups of sLNP and catechol group of pDA. siRNA reverse transfection with the pDA-sLNP-PLGA system does not exhibit cytotoxicity and induces efficient silencing of an osteogenesis inhibitor gene in human adipose-derived stem cells (hADSCs), resulting in enhanced osteogenic differentiation of hADSCs. Finally, hADSCs osteogenically committed on the pDA-sLNP-PLGA scaffolds enhanced bone formation in a mouse model of critical-sized bone defect. Therefore, the bio-inspired reverse transfection system can provide an all-in-one platform for genetic modification, differentiation, and transplantation of stem cells, simultaneously enabling both stem cell manipulation and tissue engineering.

Catechol-Functionalized Hyaluronic Acid Hydrogels Enhance Angiogenesis and Osteogenesis of Human Adipose-Derived Stem Cells in Critical Tissue Defects.

Over the last few decades, stem cell therapies have been highlighted for their potential to heal damaged tissue and aid in tissue reconstruction. However, materials used to deliver and support implanted cells often display limited efficacy, which has resulted in delaying translation of stem cell therapies into the clinic. In our previous work, we developed a mussel-inspired, catechol-functionalized hyaluronic acid (HA-CA) hydrogel that enabled effective cell transplantation due to its improved biocompatibility and strong tissue adhesiveness. The present study was performed to further expand the utility of HA-CA hydrogels for use in stem cell therapies to treat more clinically relevant tissue defect models. Specifically, we utilized HA-CA hydrogels to potentiate stem cell-mediated angiogenesis and osteogenesis in two tissue defect models: critical limb ischemia and critical-sized calvarial bone defect. HA-CA hydrogels were found to be less cytotoxic to human adipose-derived stem cells (hADSCs) in vitro compared to conventional photopolymerized HA hydrogels. HA-CA hydrogels also retained the angiogenic functionality of hADSCs and supported osteogenic differentiation of hADSCs. Because of their superior tissue adhesiveness, HA-CA hydrogels were able to mediate efficient engraftment of hADSCs into the defect regions. When compared to photopolymerized HA hydrogels, HA-CA hydrogels significantly enhanced hADSC-mediated therapeutic angiogenesis (promoted capillary/arteriole formation, improved vascular perfusion, attenuated ischemic muscle degeneration/fibrosis, and reduced limb amputation) and bone reconstruction (mineralized bone formation, enhanced osteogenic marker expression, and collagen deposition). This study proves the feasibility of using bioinspired HA-CA hydrogels as functional biomaterials for improved tissue regeneration in critical tissue defects.

Osteogenic priming of mesenchymal stem cells by chondrocyte-conditioned factors and mineralized matrix.

Transient cartilage and a mineralizing microenvironment play pivotal roles in mesenchymal cell ossification during bone formation. In order to recreate these microenvironmental cues, C3H10T1/2 murine mesenchymal stem cells (MSCs) were exposed to chondrocyte-conditioned medium (CM) and seeded onto three-dimensional mineralized scaffolds for bone regeneration. Expansion of C3H10T1/2 cells with CM resulted in enhanced expression levels of chondrogenic markers such as aggrecan, type II collagen, type X collagen, and Sox9, rather than of osteogenic genes. Interestingly, CM expansion led to reduced expression levels of osteogenic genes such as alkaline phosphatase (ALP), type I collagen, osteocalcin, and Runx2. However, CM-expanded C3H10T1/2 cells showed enhanced osteogenic differentiation as indicated by increased ALP and Alizarin Red S staining upon osteogenic factor exposure. In vivo, CM-expanded C3H10T1/2 mesenchymal cells were seeded onto mineralized scaffolds (fabricated with polydopamine and coated with simulated body fluids) and implanted into critical-sized calvarial-defect mouse models. After 8 weeks of implantation, mouse skulls were collected, and bone tissue regeneration was evaluated by micro-computed tumography and Masson's trichrome staining. In accordance with the in vitro analysis, CM-expanded C3H10T1/2 cells gave enhanced bone mineral deposition. Thus, chondrocyte-conditioned factors and a mineralized microenvironment stimulate the bone formation of MSCs.

Polydopamine-assisted osteoinductive peptide immobilization of polymer scaffolds for enhanced bone regeneration by human adipose-derived stem cells.

Immobilization of osteoinductive molecules, including growth factors or peptides, on polymer scaffolds is critical for improving stem cell-mediated bone tissue engineering. Such molecules provide osteogenesis-stimulating signals for stem cells. Typical methods used for polymeric scaffold modification (e.g., chemical conjugation or physical adsorption), however, have limitations (e.g., multistep, complicated procedures, material denaturation, batch-to-batch inconsistency, and inadequate conjugation) that diminish the overall efficiency of the process. Therefore, in this study, we report a biologically inspired strategy to prepare functional polymer scaffolds that efficiently regulate the osteogenic differentiation of human adipose-derived stem cells (hADSCs). Polymerization of dopamine (DA), a repeated motif observed in mussel adhesive protein, under alkaline pH conditions, allows for coating of a polydopamine (pDA) layer onto polymer scaffolds. Our study demonstrates that predeposition of a pDA layer facilitates highly efficient, simple immobilization of peptides derived from osteogenic growth factor (bone morphogenetic protein-2; BMP-2) on poly(lactic-co-glycolic acid) (PLGA) scaffolds via catechol chemistry. The BMP-2 peptide-immobilized PLGA scaffolds greatly enhanced in vitro osteogenic differentiation and calcium mineralization of hADSCs using either osteogenic medium or nonosteogenic medium. Furthermore, transplantation of hADSCs using pDA-BMP-2-PLGA scaffolds significantly promoted in vivo bone formation in critical-sized calvarial bone defects. Therefore, pDA-mediated catechol functionalization would be a simple and effective method for developing tissue engineering scaffolds exhibiting enhanced osteoinductivity. To the best of our knowledge, this is the first study demonstrating that pDA-mediated surface modification of polymer scaffolds potentiates the regenerative capacity of human stem cells for healing tissue defect in vivo.

The Ultra fit community mask-Toward maximal respiratory protection via personalized face fit.

Effective masking policies to prevent the spread of airborne infections depend on public access to masks with high filtration efficacy. However, poor face-fit is almost universally present in pleated multilayer disposable face masks, severely limiting both individual and community respiratory protection. We developed a set of simple mask modifications to mass-manufactured disposable masks, the most common type of mask used by the public, that dramatically improves both their personalized fit and performance in a low-cost and scalable manner. These modifications comprise a user-moldable full mask periphery wire, integrated earloop tension adjusters, and an inner flange to trap respiratory droplets. We demonstrate that these simple design changes improve quantitative fit factor by 320%, triples the level of protection against aerosolized droplets, and approaches the model efficacy of N95 respirators in preventing the community spread of COVID-19, for an estimated additional cost of less than 5 cents per mask with automated production.

Self-oxygenation of engineered living tissues orchestrates osteogenic commitment of mesenchymal stem cells.

Oxygenating biomaterials can alleviate anoxic stress, stimulate vascularization, and improve engraftment of cellularized implants. However, the effects of oxygen-generating materials on tissue formation have remained largely unknown. Here, we investigate the impact of calcium peroxide (CPO)-based oxygen-generating microparticles (OMPs) on the osteogenic fate of human mesenchymal stem cells (hMSCs) under a severely oxygen deficient microenvironment. To this end, CPO is microencapsulated in polycaprolactone to generate OMPs with prolonged oxygen release. Gelatin methacryloyl (GelMA) hydrogels containing osteogenesis-inducing silicate nanoparticles (SNP hydrogels), OMPs (OMP hydrogels), or both SNP and OMP (SNP/OMP hydrogels) are engineered to comparatively study their effect on the osteogenic fate of hMSCs. OMP hydrogels associate with improved osteogenic differentiation under both normoxic and anoxic conditions. Bulk mRNAseq analyses suggest that OMP hydrogels under anoxia regulate osteogenic differentiation pathways more strongly than SNP/OMP or SNP hydrogels under either anoxia or normoxia. Subcutaneous implantations reveal a stronger host cell invasion in SNP hydrogels, resulting in increased vasculogenesis. Furthermore, time-dependent expression of different osteogenic factors reveals progressive differentiation of hMSCs in OMP, SNP, and SNP/OMP hydrogels. Our work demonstrates that endowing hydrogels with OMPs can induce, improve, and steer the formation of functional engineered living tissues, which holds potential for numerous biomedical applications, including tissue regeneration and organ replacement therapy.

In situ microenvironment remodeling using a dual-responsive system: photodegradable hydrogels and gene activation by visible light.

A 3D microenvironment with dynamic cell-biomaterial interactions was developed using a dual-responsive system for in situ microenvironment remodeling and control of cellular function. A visible-light-responsive polymer was utilized to prepare a hydrogel with photodegradation properties, enabling in situ microenvironment remodeling. Additionally, a vascular endothelial growth factor (VEGF) gene activation unit that was responsive to the same wavelength of light was incorporated to support the potential application of the system in regenerative medicine. Following light exposure, the mechanical properties of the photodegradable hydrogel gradually deteriorated, and product analysis confirmed the degradation of the hydrogel, and thereby, 3D microenvironment remodeling. In situ microenvironment remodeling influenced stem cell proliferation and enlargement within the hydrogel. Furthermore, stem cells engineered to express light-activated VEGF and incorporated into the dual-responsive system were applied to wound healing and an ischemic hindlimb model, proving their potential application in regenerative medicine.

Fungal brain infection modelled in a human-neurovascular-unit-on-a-chip with a functional blood-brain barrier.

The neurovascular unit, which consists of vascular cells surrounded by astrocytic end-feet and neurons, controls cerebral blood flow and the permeability of the blood-brain barrier (BBB) to maintain homeostasis in the neuronal milieu. Studying how some pathogens and drugs can penetrate the human BBB and disrupt neuronal homeostasis requires in vitro microphysiological models of the neurovascular unit. Here we show that the neurotropism of Cryptococcus neoformans-the most common pathogen causing fungal meningitis-and its ability to penetrate the BBB can be modelled by the co-culture of human neural stem cells, brain microvascular endothelial cells and brain vascular pericytes in a human-neurovascular-unit-on-a-chip maintained by a stepwise gravity-driven unidirectional flow and recapitulating the structural and functional features of the BBB. We found that the pathogen forms clusters of cells that penetrate the BBB without altering tight junctions, suggesting a transcytosis-mediated mechanism. The neurovascular-unit-on-a-chip may facilitate the study of the mechanisms of brain infection by pathogens, and the development of drugs for a range of brain diseases.

A 3D culture platform enables development of zinc-binding prodrugs for targeted proliferation of ß cells.

Advances in treating ß cell loss include islet replacement therapies or increasing cell proliferation rate in type 1 and type 2 diabetes, respectively. We propose developing multiple proliferation-inducing prodrugs that target high concentration of zinc ions in ß cells. Unfortunately, typical two-dimensional (2D) cell cultures do not mimic in vivo conditions, displaying a markedly lowered zinc content, while 3D culture systems are laborious and expensive. Therefore, we developed the Disque Platform (DP)-a high-fidelity culture system where stem cell-derived ß cells are reaggregated into thin, 3D discs within 2D 96-well plates. We validated the DP against standard 2D and 3D cultures and interrogated our zinc-activated prodrugs, which release their cargo upon zinc chelation-so preferentially in ß cells. Through developing a reliable screening platform that bridges the advantages of 2D and 3D culture systems, we identified an effective hit that exhibits 2.4-fold increase in ß cell proliferation compared to harmine.

A resistance-sensing mechanical injector for the precise delivery of liquids to target tissue.

The precision of the delivery of therapeutics to the desired injection site by syringes and hollow needles typically depends on the operator. Here, we introduce a highly sensitive, completely mechanical and cost-effective injector for targeting tissue reliably and precisely. As the operator pushes the syringe plunger, the injector senses the loss-of-resistance on encountering a softer tissue or a cavity, stops advancing the needle and delivers the payload. We demonstrate that the injector can reliably deliver liquids to the suprachoroidal space-a challenging injection site that provides access to the back of the eye-for a wide range of eye sizes, scleral thicknesses and intraocular pressures, and target sites relevant for epidural injections, subcutaneous injections and intraperitoneal access. The design of this simple and effective injector can be adapted for a broad variety of clinical applications.

Ferritin nanoparticles for improved self-renewal and differentiation of human neural stem cells.

BACKGROUND: Biomaterials that promote the self-renewal ability and differentiation capacity of neural stem cells (NSCs) are desirable for improving stem cell therapy to treat neurodegenerative diseases. Incorporation of micro- and nanoparticles into stem cell culture has gained great attention for the control of stem cell behaviors, including proliferation and differentiation. METHOD: In this study, ferritin, an iron-containing natural protein nanoparticle, was applied as a biomaterial to improve the self-renewal and differentiation of NSCs and neural progenitor cells (NPCs). Ferritin nanoparticles were added to NSC or NPC culture during cell growth, allowing for incorporation of ferritin nanoparticles during neurosphere formation. RESULTS: Compared to neurospheres without ferritin treatment, neurospheres with ferritin nanoparticles showed significantly promoted self-renewal and cell-cell interactions. When spontaneous differentiation of neurospheres was induced during culture without mitogenic factors, neuronal differentiation was enhanced in the ferritin-treated neurospheres. CONCLUSIONS: In conclusion, we found that natural nanoparticles can be used to improve the self-renewal ability and differentiation potential of NSCs and NPCs, which can be applied in neural tissue engineering and cell therapy for neurodegenerative diseases.

Electrospun Silk Fibroin Nanofibrous Scaffolds with Two-Stage Hydroxyapatite Functionalization for Enhancing the Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

The development of functional scaffolds with improved osteogenic potential is important for successful bone formation and mineralization in bone tissue engineering. In this study, we developed a functional electrospun silk fibroin (SF) nanofibrous scaffold functionalized with two-stage hydroxyapatite (HAp) particles, using mussel adhesive-inspired polydopamine (PDA) chemistry. HAp particles were first incorporated into SF scaffolds during the electrospinning process, and then immobilized onto the electrospun SF nanofibrous scaffolds containing HAp via PDA-mediated adhesive chemistry. We obtained two-stage HAp-functionalized SF nanofibrous scaffolds with improved mechanical properties and capable of providing a bone-specific physiological microenvironment. The developed scaffolds were tested for their ability to enhance the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) in vitro and repair bone defect in vivo. To boost their ability for bone repair, we genetically modified hADMSCs with the transcriptional coactivator with PDZ-binding motif (TAZ) via polymer nanoparticle-mediated gene delivery. TAZ is a well-known transcriptional modulator that activates the osteogenic differentiation of mesenchymal stem cells (MSCs). Two-stage HAp-functionalized SF scaffolds significantly promoted the osteogenic differentiation of TAZ-transfected hADMSCs in vitro and enhanced mineralized bone formation in a critical-sized calvarial bone defect model. Our study shows the potential utility of SF scaffolds with nanofibrous structures and enriched inorganic components in bone tissue engineering.

Biodegradable Nerve Guidance Conduit with Microporous and Micropatterned Poly(lactic-co-glycolic acid)-Accelerated Sciatic Nerve Regeneration.

An innovative technique combining capillary force lithography and phase separation method in one step is applied to fabricate artificial nerve guidance conduit (NGC) for peripheral nerve regeneration. Biodegradable porous, patterned NGC (PP-NGC) using poly(lactic-co-glycolic acid) is fabricated. It has micro-grooves and microporosity on the inner surface to promote axonal outgrowth and to enhance permeability for nutrient exchange. In this study, it is confirmed that the inner surface of micro-grooves can modulate neurite orientation and length of mouse neural stem cell compared to porous flat NGC (PF-NGC) in vitro. Coating with 3,4-dihydroxy-l-phenylalanine (DOPA) facilitates the hydrophilic inner surface of PF- and PP-NGCs via bioinspired catechol chemistry. For in vivo study, PF-NGC and PP-NGC coated with or without DOPA are implanted in the 10 mm sciatic nerve defect margins between proximal and distal nerves in rats. Especially, PP-NGC coated with DOPA shows higher sciatic function index score, onset-to-peak amplitude, and muscle fiber diameter compared to other groups. The proposed hybrid-structured NGC not only can serve as a design for functional NGC without growth factor but also can be used in clinical application for peripheral nerve regeneration.

In Situ Bone Tissue Engineering With an Endogenous Stem Cell Mobilizer and Osteoinductive Nanofibrous Polymeric Scaffolds.

Classical bone tissue engineering involves the use of culture-expanded cells and scaffolds to produce tissue constructs for transplantation. Despite promising results, clinical adoption of these constructs has been limited due to various drawbacks, including extensive cell expansion steps, low cell survival rate upon transplantation, and the possibility of immuno-rejection. To bypass the ex vivo cell culture and transplantation process, the regenerative capacity of the host is exploited by mobilizing endogenous stem cells to the site of injury. Systemic injection of substance P (SP) induce mobilization of CD29+ CD105+ CD45- cells from bone marrow and enhance bone tissue regeneration in a critical-sized calvarial bone defect model. To provide an appropriate environment for endogenous stem cells to survive and differentiate into osteogenic lineage cells, electrospun nanofibrous polycaprolactone (PCL) scaffolds are functionalized with hydroxyapatite (HA) particles via a polydopamine (PDA) coating to create highly osteoinductive PCL-PDA-HA scaffolds that are implanted in defects. The combination of the PCL-PDA-HA scaffold and SP treatment enhance in situ bone tissue formation in defects. Thus, this in situ bone regeneration strategy, which combines recruitment of endogenous stem cells from the bone marrow to defective sites and implantation of a highly biocompatible and osteoinductive cell-free scaffold system, has potential as an effective therapeutic in regenerative medicine.

Photoactive Poly(3-hexylthiophene) Nanoweb for Optoelectrical Stimulation to Enhance Neurogenesis of Human Stem Cells.

Optoelectrical manipulation has recently gained attention for cellular engineering; however, few material platforms can be used to efficiently regulate stem cell behaviors via optoelectrical stimulation. In this study, we developed nanoweb substrates composed of photoactive polymer poly(3-hexylthiophene) (P3HT) to enhance the neurogenesis of human fetal neural stem cells (hfNSCs) through photo-induced electrical stimulation. METHODS: The photoactive nanoweb substrates were fabricated by self-assembled one-dimensional (1D) P3HT nanostructures (nanofibrils and nanorods). The hfNSCs cultured on the P3HT nanoweb substrates were optically stimulated with a green light (539 nm) and then differentiation of hfNSCs on the substrates with light stimulation was examined. The utility of the nanoweb substrates for optogenetic application was tested with photo-responsive hfNSCs engineered by polymer nanoparticle-mediated transfection of an engineered chimeric opsin variant (C1V1)-encoding gene. RESULTS: The nanoweb substrates provided not only topographical stimulation for activating focal adhesion signaling of hfNSCs, but also generated optoelectrical stimulation via photochemical and charge-transfer reactions upon exposure to 539 nm wavelength light, leading to significantly enhanced neuronal differentiation of hfNSCs. The optoelectrically stimulated hfNSCs exhibited mature neuronal phenotypes with highly extended neurite formation and functional neuron-like electrophysiological features of sodium currents and action potentials. Optoelectrical stimulation with 539 nm light simultaneously activated both C1V1-modified hfNSCs and nanoweb substrates, which upregulated the expression and activation of voltage-gated ion channels in hfNSCs and further increased the effect of photoactive substrates on neuronal differentiation of hfNSCs. CONCLUSION: The photoactive nanoweb substrates developed in this study may serve as platforms for producing stem cell therapeutics with enhanced neurogenesis and neuromodulation via optoelectrical control of stem cells.

Electroconductive nanoscale topography for enhanced neuronal differentiation and electrophysiological maturation of human neural stem cells.

Biophysical cues, such as topography, and electrical cues can provide external stimulation for the promotion of stem cell neurogenesis. Here, we demonstrate an electroconductive surface nanotopography for enhancing neuronal differentiation and the functional maturation of human neural stem cells (hNSCs). The electroconductive nanopatterned substrates were prepared by depositing a thin layer of titanium (Ti) with nanograting topographies (150 to 300 nm groove/ridge, the thickness of the groove - 150 µm) onto polymer surfaces. The Ti-coated nanopatterned substrate (TNS) induced cellular alignment along the groove pattern via contact guidance and promoted focal adhesion and cytoskeletal reorganization, which ultimately led to enhanced neuronal differentiation and maturation of hNSCs as indicated by significantly elevated neurite extension and the upregulated expression of the neuronal markers Tuj1 and NeuN compared with the Ti-coated flat substrate (TFS) and the nanopatterned substrate (NS) without Ti coating. Mechanosensitive cellular events, such as ß1-integrin binding/clustering and myosin-actin interaction, and the Rho-associated protein kinase (ROCK) and mitogen-activated protein kinase/extracellular signal regulated kinase (MEK-ERK) pathways, were found to be associated with enhanced focal adhesion and neuronal differentiation of hNSCs by the TNS. Among the neuronal subtypes, differentiation into dopaminergic and glutamatergic neurons was promoted on the TNS. Importantly, the TNS increased the induction rate of neuron-like cells exhibiting electrophysiological properties from hNSCs. Finally, the application of pulsed electrical stimulation to the TNS further enhanced neuronal differentiation of hNSCs due probably to calcium channel activation, indicating a combined effect of topographical and electrical cues on stem cell neurogenesis, which postulates the novelty of our current study. The present work suggests that an electroconductive nanopatterned substrate can serve as an effective culture platform for deriving highly mature, functional neuronal lineage cells from stem cells.

Enhanced Self-Renewal and Accelerated Differentiation of Human Fetal Neural Stem Cells Using Graphene Oxide Nanoparticles.

Graphene oxide (GO) has received increasing attention in bioengineering fields due to its unique biophysical and electrical properties, along with excellent biocompatibility. The application of GO nanoparticles (GO-NPs) to engineer self-renewal and differentiation of human fetal neural stem cells (hfNSCs) is reported. GO-NPs added to hfNSC culture during neurosphere formation substantially promote cell-to-cell and cell-to-matrix interactions in neurospheres. Accordingly, GO-NP-treated hfNSCs show enhanced self-renewal ability and accelerated differentiation compared to untreated cells, indicating the utility of GO in developing stem cell therapies for neurogenesis.