The Regulatory Functionality of Exosomes Derived from hUMSCs in 3D Culture for Alzheimer's Disease Therapy.

Reducing amyloid-ß (Aß) accumulation could be a potential therapeutic approach for Alzheimer's disease (AD). Particular functional biomolecules in exosomes vested by the microenvironment in which the original cells resided can be transferred to recipient cells to improve pathological conditions. However, there are few reports addressing whether exosomes derived from cells cultured on scaffolds with varying dimension can reduce Aß deposition or ameliorate cognitive decline for AD therapy. Herein, both 3D graphene scaffold and 2D graphene film are used as the matrix for human umbilical cord mesenchymal stem cell culture, from which the supernatants are obtained to isolate exosomes. The levels of 195 kinds of miRNAs and proteins, including neprilysin, insulin-degrading enzyme and heat shock protein 70, in 3D-cultured exosomes (3D-Exo) are dramatically different from those obtained from 2D culture. Hence, 3D-Exo could up-regulate the expression of α-secretase and down-regulate the ß-secretase to reduce Aß production in both AD pathology cells and transgenic mice, through their special cargo. With rescuing Aß pathology, 3D-Exo exerts enhanced therapeutic effects on ameliorating the memory and cognitive deficits in AD mice. These findings provide a novel clinical application for scaffold materials and functional exosomes derived from stem cells.

Bone marrow-inspired hydrogel/graphene composite scaffolds to support in vitro expansion of hematopoietic stem cells.

Hematopoietic stem cell (HSC) expansion offers a key strategy to address the source limitation and donor shortages of HSCs for the treatment of various blood disorders. Specific remodeling of the complex bone marrow microenvironment that contributes to efficient in vitro expansion of HSCs remains challenging. Here, inspired by the regions with different stiffness levels in the bone marrow niche, a three dimensional (3D) bone marrow-mimicking composite scaffold created based on gelatin-hyaluronic acid (Gel-HA) hydrogels and graphene foams (GFs) was engineered to support the in vitro expansion of HSCs. The composite scaffold was prepared by forming a photo-cross-linked Gel-HA hydrogel surrounding the GF. The "soft" Gel-HA hydrogel and "stiff" GF replicate the structure and stiffness of the vascular niche and endosteal niche in the bone marrow, respectively. Furthermore, HSCs cultured in the Gel-HA/GF scaffold proliferated well and retained the CD34+CD38- immunophenotype and pluripotency, suggesting that the Gel-HA/GF composite scaffold supported the in vitro expansion of HSCs, maintaining the primitive phenotype and the ability to differentiate into functional blood cells. Thus, the hydrogel/graphene composite scaffold offers a means of facilitating HSC expansion through structurally and mechanically mimicking bone marrow niches, demonstrating great promise for HSC transplantation.

Annular Conductive Hydrogel-Mediated Wireless Electrical Stimulation for Augmenting Neurogenesis.

Electrical stimulation (ES) has a remarkable capacity to regulate neuronal differentiation and neurogenesis in the treatment of various neurological diseases. However, wired devices connected to the stimulating electrode and the mechanical mismatch between conventional rigid electrodes and soft tissues restrict their motion and cause possible infections, thereby limiting their clinical utility. An approach integrating the advantages of wireless techniques and soft hydrogels provides new insights into ES-induced nerve regeneration. Herein, a flexible and implantable wireless ES-responsive electrode based on an annular gelatin methacrylate-polyaniline (Gel/Pani) hydrogel is fabricated and used as a secondary coil to achieve wireless ES via electromagnetic induction in the presence of a primary coil. The Gel/Pani hydrogels exhibit favorable biocompatibility, biodegradability, conductivity, and compression resistance. The annular electrode of the Gel/Pani conductive hydrogel (AECH) supports neural stem cell growth, while the applied wireless ES facilitates neuronal differentiation and the formation of functional neural networks in vitro. Furthermore, AECH is implanted in vivo in rats with ischemic stroke and the results reveal that AECH-mediated wireless ES significantly ameliorates brain impairment and neurological function by activating endogenous neurogenesis. This novel flexible hydrogel system addresses wireless stimulation and implantable technical challenges, holding great potential for the treatment of neurodegenerative diseases.

Crocetin Regulates Functions of Neural Stem Cells to Generate New Neurons for Cerebral Ischemia Recovery.

Many neurons undergo apoptosis after ischemic stroke. In the brain, neurogenesis has the potential for neuronal replacement and can be activated by external conditions to repair the injury. Crocetin (CRO), naturally extracted from the plant saffron, acts as a neuroprotective agent for ischemic stroke. However, the underlying mechanism remains unknown. In this work, the effect of CRO on neural stem cell (NSC) behaviors and subventricular zone neurogenesis is investigated. Initially, NSCs are incubated with different concentrations of CRO to detect the cell proliferation and differentiation in vitro. Second, ischemic stroke induced rats are treated with CRO using nimodipine (NMDP) as a comparison. The behavioral functions, infarcted volume, and apoptotic Nissl bodies of rats are noticeably improved after CRO-treatment, comparable to those of NMDP. In addition, the increased regional cerebral blood flow and promoted neuronal differentiation are achieved by CRO-treatment. Brain tissue examination shows significantly increased neuronal regeneration in the focal ischemic injury area. Meanwhile, the length of neurites is prolonged, indicating that CRO could potentially promote neurite extension to enhance cell-cell communication. These findings demonstrate that CRO facilitated the neuronal differentiation of NSCs by activating subventricular zone neurogenesis in damaged cortex and striatum sites to repair ischemic stroke.

A precise design strategy for a cell-derived extracellular matrix based on CRISPR/Cas9 for regulating neural stem cell function.

The extracellular matrix (ECM) is a natural microenvironment pivotal for stem cell survival, as well as proliferation, differentiation and metastasis, composed of a variety of biological molecular complexes secreted by resident cells in tissues and organs. Heparan sulfate proteoglycan (HSPG) is a type of ECM protein that contains one or more covalently attached heparan sulfate chains. Heparan sulphate chains have high affinity with growth factors, chemokines and morphogens, acting as cytokine-binding domains of great importance in development and normal physiology. Herein, we constructed endogenous HSPG2 overexpression in mouse embryonic fibroblasts based on the CRISPR/Cas9 synergistic activation mediator system and then fabricated a cell-derived HSPG2 functional ECM (ECMHSPG2). The ECMHSPG2 is capable of enriching basic fibroblast growth factor (bFGF), which binds more strongly than the negative control ECM. With a growing bFGF concentration, ECMHSPG2 could better maintain neural stem cell (NSCs) stemness and promote NSC proliferation and differentiation in culture. These findings provide a precise design strategy for producing a specific cell-derived ECM for biomaterials in research and regenerative medicine.

Injectable and Degradable POSS-Polyphosphate-Polysaccharide Hybrid Hydrogel Scaffold for Cartilage Regeneration.

The limited self-repair capacity of articular cartilage has motivated the development of stem cell therapy based on artificial scaffolds that mimic the extracellular matrix (ECM) of cartilage tissue. In view of the specificity of articular cartilage, desirable tissue adhesiveness and stable mechanical properties under cyclic mechanical loads are critical for cartilage scaffolds. Herein, we developed an injectable and degradable organic-inorganic hybrid hydrogel as a cartilage scaffold based on polyhedral oligomeric silsesquioxane (POSS)-cored polyphosphate and polysaccharide. Specifically, acrylated 8-arm star-shaped POSS-poly(ethyl ethylene phosphate) (POSS-8PEEP-AC) was synthesized and cross-linked with thiolated hyaluronic acid (HA-SH) to form a degradable POSS-PEEP/HA hydrogel. Incorporation of POSS in the hydrogel increased the mechanical properties. The POSS-PEEP/HA hydrogel showed enzymatic biodegradability and favorable biocompatibility, supporting the growth and differentiation of human mesenchymal stem cells (hMSCs). The chondrogenic differentiation of encapsulated hMSCs was promoted by loading transforming growth factor-ß3 (TGF-ß3) in the hydrogel. In addition, the injectable POSS-PEEP/HA hydrogel was capable of adhering to rat cartilage tissue and resisting cyclic compression. Furthermore, in vivo results revealed that the transplanted hMSCs encapsulated in the POSS-PEEP/HA hydrogel scaffold significantly improved cartilage regeneration in rats, while the conjugation of TGF-ß3 achieved a better therapeutic effect. The present work demonstrated the potential of the injectable, biodegradable, and mechanically enhanced POSS-PEEP/HA hybrid hydrogel as a scaffold biomaterial for cartilage regeneration.

High-efficiency brain-targeted intranasal delivery of BDNF mediated by engineered exosomes to promote remyelination.

The regeneration of myelin sheaths is the ultimate goal of the treatment of demyelination disease, including multiple sclerosis (MS). However, current drugs for MS mainly target the immune system and can only slow down the disease development and do not promote the differentiation of oligodendrocyte precursor cells (OPCs) abundant in the myelin injury region into mature oligodendrocytes to form a new myelin sheath. Brain-derived neurotrophic factor (BDNF) plays an important role in the regulation of OPC proliferation and differentiation into mature oligodendrocytes. Exosomes, a kind of nanoscale membrane vesicle secreted by cells, can be used as potential therapeutic drug delivery vectors for central nervous system diseases. Here, brain-targeted modification and BDNF intracellular-loaded exosomes were produced through engineering HEK293T cells, which can promote the differentiation of OPCs into mature oligodendrocytes in vitro. The intranasal administration of the brain-targeted engineered exosome-mediated BDNF was a highly effective delivery route to the brain and had a significant therapeutic effect on remyelination and motor coordination ability improvement in demyelination model mice. The combination of intranasal administration with brain-targeted and BDNF-loaded designed exosomes provides a strategy for efficient drug delivery and treatment of central nervous system diseases.

Specific anti-glioma targeted-delivery strategy of engineered small extracellular vesicles dual-functionalised by Angiopep-2 and TAT peptides.

Glioma is one of the primary malignant brain tumours in adults, with a poor prognosis. Pharmacological reagents targeting glioma are limited to achieve the desired therapeutic effect due to the presence of blood-brain barrier (BBB). Effectively crossing the BBB and specifically targeting to the brain tumour are the major challenge for the glioma treatments. Here, we demonstrate that the well-defined small extracellular vesicles (sEVs) with dual-targeting drug delivery and cell-penetrating functions, modified by Angiopep-2 and trans-activator of transcription peptides, enable efficient and specific chemotherapy for glioma. The high efficiency of engineered sEVs in targeting BBB and glioma was assessed in both monolayer culture cells and BBB model in vitro, respectively. The observed high targeting efficiency was re-validated in subcutaneous tumour and orthotopic glioma mice models. After loading the doxorubicin into dual-modified functional sEVs, this specific dual-targeting delivery system could cross the BBB, reach the glioma, and penetrate the tumour. Such a mode of drug delivery significantly improved more than 2-fold survival time of glioma mice with very few side effects. In conclusion, utilization of the dual-modified sEVs represents a unique and efficient strategy for drug delivery, holding great promise for the treatments of central nervous system diseases.

Cell-derived extracellular matrix enhanced by collagen-binding domain-decorated exosomes to promote neural stem cells neurogenesis.

Enhancing neurogenesis of neural stem cells (NSCs) is crucial in stem cell therapy for neurodegenerative diseases. Within the extracellular microenvironment, extracellular matrix (ECM) plays a pivotal role in modulating cell behaviors. However, a single ECM biomaterial is not sufficient to establish an ideal microenvironment. As multifunctional nanocarriers, exosomes display tremendous advantages for the treatments of various diseases. Herein, collagen binding domain peptide-modified exosomes (CBD-Exo) were obtained from the SH-SY5Y cell line infected with lentivirus particles encoding CBD-lysosome associated membrane glycoprotein 2b (CBD-Lamp2b) to improve the binding efficiency of exosomes and ECM. An exosomes-functionalized ECM (CBD-Exo/ECM) was then constructed via the interaction between CBD and collagen in ECM. Then, CBD-Exo/ECM was employed as a carrier for NSCs culture. The results showed that CBD-Exo/ECM can support the neurogenesis of NSCs with the percentage of proliferation marker EdU-positive (35.8% ± 0.47% vs 21.9% ± 2.32%) and neuron maker Tuj-1-positive (55.8% ± 0.47% vs 30.6% ± 2.62%) were both significantly increased in the exosomes-functionalized ECM system. This exosomes-functionalized ECM was capable to promote the cell proliferation and accelerate neuronal differentiation of NSCs, providing a potential biomedical material for stem cell application in tissue engineering and regenerative medicine.

Strategy for Designing a Cell Scaffold to Enable Wireless Electrical Stimulation for Enhanced Neuronal Differentiation of Stem Cells.

Electrical stimulation (ES) offers significant advantages in modulating the behavior of stem cells on conductive scaffolds for neural tissue engineering. However, it is necessary to realize wireless ES to avoid the use of external wires in tissues. Thus, herein, a strategy is reported to develop a stem cell scaffold that allows wireless ES. A conductive annular graphene substrate is designed and grown by chemical vapor deposition; this substrate is used as a secondary coil to achieve wireless ES via electromagnetic induction in the presence of a primary coil. The substrate shows excellent biocompatibility for the culture of neural stem cells (NSCs). The results indicate that the applied wireless ES enhances neuronal differentiation, facilitates the formation of neurites, and does not substantially affect the viability and stemness maintenance of NSCs. Collectively, this system provides a strategy for achieving synergy between wireless ES and conductive scaffolds for neural regenerative medicine, which can be further utilized for the regeneration of other tissues.