Therapeutic Efficacy-Potentiated and Diseased Organ-Targeting Nanovesicles Derived from Mesenchymal Stem Cells for Spinal Cord Injury Treatment

Human mesenchymal stem cell (hMSC)-derived exosomes have been spotlighted as a promising therapeutic agent for cell-free regenerative medicine. However, poor organ-targeting ability and insufficient therapeutic efficacy of systemically injected hMSC-exosomes were identified as critical limitations for their further applications. Therefore, in this study we fabricated iron oxide nanoparticle (IONP)–incorporated exosome-mimetic nanovesicles (NV-IONP) from IONP-treated hMSCs and evaluated their therapeutic efficacy in a clinically relevant model for spinal cord injury. Compared to exosome-mimetic nanovesicles (NV) prepared from untreated hMSCs, NV-IONP not only contained IONPs which act as a magnet-guided navigation tool but also carried greater amounts of therapeutic growth factors that can be delivered to the target cells. The increased amounts of therapeutic growth factors inside NV-IONP were attributed to IONPs that are slowly ionized to iron ions which activate the JNK and c-Jun signaling cascades in hMSCs. In vivo systemic injection of NV-IONP with magnetic guidance significantly increased the amount of NV-IONP accumulating in the injured spinal cord. Accumulated NV-IONP enhanced blood vessel formation, attenuated inflammation and apoptosis in the injured spinal cord, and consequently improved spinal cord function. Taken together, these findings highlight the development of therapeutic efficacy-potentiated extracellular nanovesicles and demonstrate their feasibility for repairing injured spinal cord

Dual Roles of Graphene Oxide To Attenuate Inflammation and Elicit Timely Polarization of Macrophage Phenotypes for Cardiac Repair

Development of localized inflammatory environments by M1 macrophages in the cardiac infarction region exacerbates heart failure after myocardial infarction (MI). Therefore, the regulation of inflammation by M1 macrophages and their timely polarization toward regenerative M2 macrophages suggest an immunotherapy. Particularly, controlling cellular generation of reactive oxygen species (ROS), which cause M1 differentiation, and developing M2 macrophage phenotypes in macrophages propose a therapeutic approach. Previously, stem or dendritic cells were used in MI for their anti-inflammatory and cardioprotective potentials and showed inflammation modulation and M2 macrophage progression for cardiac repair. However, cell-based therapeutics are limited due to invasive cell isolation, time-consuming cell expansion, labor-intensive and costly <i>ex vivo</i> cell manipulation, and low grafting efficiency. Here, we report that graphene oxide (GO) can serve as an antioxidant and attenuate inflammation and inflammatory polarization of macrophages <i>via</i> reduction in intracellular ROS. In addition, GO functions as a carrier for interleukin-4 plasmid DNA (IL-4 pDNA) that propagates M2 macrophages. We synthesized a macrophage-targeting/polarizing GO complex (MGC) and demonstrated that MGC decreased ROS in immune-stimulated macrophages. Furthermore, DNA-functionalized MGC (MGC/IL-4 pDNA) polarized M1 to M2 macrophages and enhanced the secretion of cardiac repair-favorable cytokines. Accordingly, injection of MGC/IL-4 pDNA into mouse MI models attenuated inflammation, elicited early polarization toward M2 macrophages, mitigated fibrosis, and improved heart function. Taken together, the present study highlights a biological application of GO in timely modulation of the immune environment in MI for cardiac repair. Current therapy using off-the-shelf material GO may overcome the shortcomings of cell therapies for MI

<i>In Vivo</i> Bioluminescence Imaging for Prolonged Survival of Transplanted Human Neural Stem Cells Using 3D Biocompatible Scaffold in Corticectomized Rat Model

<div><p>Stem cell-based treatment of traumatic brain injury has been limited in its capacity to bring about complete functional recovery, because of the poor survival rate of the implanted stem cells. It is known that biocompatible biomaterials play a critical role in enhancing survival and proliferation of transplanted stem cells via provision of mechanical support. In this study, we noninvasively monitored <i>in vivo</i> behavior of implanted neural stem cells embedded within poly-l-lactic acid (PLLA) scaffold, and showed that they survived over prolonged periods in corticectomized rat model. Corticectomized rat models were established by motor-cortex ablation of the rat. F3 cells expressing enhanced firefly luciferase (F3-effLuc) were established through retroviral infection. The F3-effLuc within PLLA was monitored using IVIS-100 imaging system 7 days after corticectomized surgery. F3-effLuc within PLLA robustly adhered, and gradually increased luciferase signals of F3-effLuc within PLLA were detected in a day dependent manner. The implantation of F3-effLuc cells/PLLA complex into corticectomized rats showed longer-lasting luciferase activity than F3-effLuc cells alone. The bioluminescence signals from the PLLA-encapsulated cells were maintained for 14 days, compared with 8 days for the non-encapsulated cells. Immunostaining results revealed expression of the early neuronal marker, Tuj-1, in PLLA-F3-effLuc cells in the motor-cortex-ablated area. We observed noninvasively that the mechanical support by PLLA scaffold increased the survival of implanted neural stem cells in the corticectomized rat. The image-guided approach easily proved that scaffolds could provide supportive effect to implanted cells, increasing their viability in terms of enhancing therapeutic efficacy of stem-cell therapy.</p></div

Schematic representation of the procedure for <i>in vivo</i> optical imaging.

<p>(A) The protocol is for <i>in vivo</i> monitoring of F3-effLuc cells implanted in a corticectomized rat model. The motor cortex region of the Sprague-Dawley rat brain was surgically removed at the given coordinates and after 7 days, the rats were transplanted with F3-effLuc cells alone or F3-effLuc/PLLA scaffold complexes, and then administered cyclosporine A everyday. The grafted cells were monitored at 0, 1, 3, 5, 8, 11, and 14 days using a bioluminescence-imaging device. At the end of the implant period, histological analyses were performed using hematoxylin and eosin (H&E) staining and immunohistochemistry. (B) Behavior tests were performed 7 days after motor cortex ablation. The traumatic brain injury (TBI) models were evaluated by forelimb placing tests and whisker tactile tests in normal and corticectomized rats (n = 10).</p

<i>In vivo</i> bioluminescence imaging of the implanted F3-effLuc/PLLA scaffold in a corticectomized rat model.

<p>(A) After F3-effLuc cells were incubated within the PLLA scaffold for 2 hr, the cell/scaffold complex was implanted into the ablated motor cortex area of the rat brain. Firefly luciferase bioluminescence imaging was performed over 14 days. The prolonged luminescence signals in F3-effLuc cells within the PLLA scaffold were clearly visualized in the ablated area. (B) Quantitative ROI analysis showed significantly enhanced survival duration for F3-effLuc cells within the PLLA scaffold (n = 6). P value, * <0.005.</p

<i>In vitro</i> proliferative effect in F3-effLuc cells within the PLLA scaffold.

<p>(A) Scanning electron microscope (SEM) analysis was conducted to confirm adhesion of F3-effLuc cells to the PLLA scaffold. SEM images showed that F3-effLuc cells were stably attached onto the microfibers of the PLLA scaffold. (B) The luciferase intensity was quantified after F3-effLuc cells were incubated with the sterile PLLA scaffold. F3-effLuc cells incorporated within the PLLA scaffold were stably proliferated at 10 days.</p

Validation of stem cell characteristics in F3 cells and F3-effLuc cells.

<p>Flow cytometric analysis showed F3 and F3-effLuc cells are positive for the stem cell surface marker, (a) CD44, and the intracellular marker s, (b) Nestin, (c) Ki67, (d) Sox1, and (e) Sox2.</p

<i>In vitro</i> luciferase reporter activity in F3-effLuc cells.

<p>(A) Retroviral construct that contains the <i>effLuc</i> gene and Thy1.1 (CD90.1), linked with an IRES (internal ribosomal entry site). (B) Magnetic-activated cell sorting (MACS) was performed to collect the F3-effLuc cells. Flow activated cell sorting (FACS) analysis showed that more than 90% of cells were successively transfected with the <i>effLuc</i> vector. (C) The luciferase activity (n = 3) of F3-effLuc cells cultured in a 96-well plate were measured using an IVIS-100 optical imaging device. Firefly luciferase activity continuously increased in F3-effLuc cells in proportion to cell number, and (D) quantitative analysis showed a linear relationship between the cell number and bioluminescence signals.</p

Immunohistochemistry results for PLLA-encapsulated F3-effLuc cells implanted in a corticectomized rat brain.

<p>(A) Hematoxylin and eosin (H&E) staining was performed on the corticectomized rat brains transplanted with the F3-effLuc/PLLA scaffold complex. Slices of the fixed brain were stained with hematoxylin and eosin to examine the presence of transplanted cells on the PLLA microfibers. (B) Confocal fluorescence images of the transplanted region (purple) revealed luciferase expression (green) in F3-effLuc cells was partially co-localized with the expression of Tuj1, a neuron-specific marker (red). Nuclei were visualized with DAPI (blue). Scale bars represent 20 µm. No luciferase expression was observed in F3 cell only-transplanted rat model.</p