Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/NanoLett
Magnetic Control of Axon Navigation in Reprogrammed Neurons Yoonhee Jin,§ Jung-uk Lee,†,∥ Eunna Chung,†,∥ Kisuk Yang,§ Jin Kim,§ Ji-wook Kim,†,∥ Jong Seung Lee,§ Ann-Na Cho,§ Taekyu Oh,†,‡ Jae-Hyun Lee,†,‡ Seung-Woo Cho,*,†,‡,§ and Jinwoo Cheon*,†,‡,∥ †
Center for Nanomedicine, Institute for Basic Science (IBS), Seoul 03722, Republic of Korea Graduate Program of Nano Biomedical Engineering (Nano BME), Yonsei-IBS Institute, Yonsei University, Seoul 03722, Republic of Korea § Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea ∥ Department of Chemistry, Yonsei University, Seoul 03722, Republic of Korea
Downloaded via NOTTINGHAM TRENT UNIV on August 29, 2019 at 02:16:51 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
S Supporting Information *
ABSTRACT: 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. KEYWORDS: magnetic nanoparticles, receptor clustering, induced neuronal cells, axon guidance, neural network
C
involves certain limitations stemming from genetic manipulations with viral vector injections and difficulties in the optical penetration through target tissues.7,8 As another type of method, magnetic approaches offer new opportunities to deliver physical cues of mechanical force or heat to the target tissue at the single or subcellular level.9−13 For example, the magnetic activation of angiogenetic and apoptotic pathways in endothelial and cancer cells were demonstrated in vitro and in vivo.14,15 Also, Arg-Gly-Asp (RGD) peptide conjugated MNPs were shown to activate mechanotransduction pathways and to regulate stem cell adhesion, spreading, and differentiation.16,17 Similarly, a magnetogenetic approach was reported for the subcellular-level control of mechanoreceptors, such as RhoGTPase, FcεRI, and Notch receptors, with applied forces in the pico-Newton range.9,18,19 In this study, we introduce the remote magnetic control of neurite outgrowth in induced neuronal (iN) cells to achieve functional connections with pre-existing neurons. The formation of neural circuits depends on the coordination of guidance cues via the activation of membrane protein receptors and intracellular signal transduction cascades, all of which
ell therapy has emerged as an effective treatment for neural regeneration and behavioral recovery in neurodegenerative disorders.1,2 This approach can be potentiated by engineering strategies to improve the survival and engraftment of transplanted cells in defective regions. Currently, achieving proper interactions between grafted cells and host neurons with desired connectivity remains a critical step.1,2 In this context, the ability to direct axons from grafted cells toward host cells and to create connections is necessary. Conventional approaches to direct axonal regrowth and elongation across nerve lesions have involved biochemical guiding cues, such as molecules capable of orienting and migrating cells (e.g., netrins, ephrins, and semaphorins) and factors influencing neuronal growth and functions (e.g., growth factors and neurotransmitters).3 However, limited spatial control in axon guidance with biochemical-molecule-based approaches impedes the efficacy of synaptic guidance to reconnect neurons. As the counterparts of biochemical cues, physical cues are potentially beneficial for modulating biological behaviors in a spatiotemporally controlled manner.4,5 Recent progress in optogenetics has opened new opportunities for the activation and deactivation of cell signals at the single-cell level with millisecond-level precision.6 Optogenetics has benefited neuroscience and oncology, for example, by providing a better understanding of cellular circuitry and alternative clinical approaches for treating diseased lesions. However, it often © XXXX American Chemical Society
Received: July 6, 2019 Revised: August 8, 2019
A
DOI: 10.1021/acs.nanolett.9b02756 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters
Figure 1. Schematic illustration of neurite guidance, characterization of DCC-MNPs, and DCC-MNP clustering under a magnetic field. (a−c) Scheme of binding of DCC antibody-conjugated MNPs (DCC-MNPs) to iN cells expressing the DCC receptor (a), clustering of DCC-MNPs bound to iN cells under a magnetic field for neurite extension (b), and functional network formation between treated iN cells and host neurons via guidance of the neurite growth of iN cells (c). (d) Transmission electron microscopy image of the MNP core (scale bar = 20 nm). (e) Preparation of DCC-MNPs. (f) Size of MNPs and DCC-MNPs determined by dynamic light scattering analysis. (g) Fluorescence microscopy image of iN cells bound to Fl-DCC-MNPs to observe the distribution of DCC clusters under the gradient magnetic field (GMF) and uniform magnetic field (UMF) compared with w/o magnetic field and no treatment (NT) group (scale bar = 10 μm). (h) Schematic of the measurement of the directionality of clustered particles. The angle theta (θ) was defined as the angle between the external magnetic field direction and clustered magnetic nanoparticles relative to the center of the cell nucleus. (i) Theta (θ) values of magnetic nanoparticle clusters under the gradient and uniform magnetic fields (N = 15, respectively). (j) Cos θ values of magnetic nanoparticle clusters under the GMF and UMF.
axonal outgrowth for improved synaptic interactions with host tissue is critical. For the clustering of DCC receptors, superparamagnetic 13 nm zinc-doped iron oxide MNPs (Figure 1d) with a high saturation magnetization value (∼130 emu/gmetal atom) were used. The MNPs were then coated with silica and protein A (ProA-MNPs), followed by conjugation with DCC antibody (DCC Ab) (Figure 1e). Fluorescein (Fl) was also conjugated to ProA for fluorescence imaging of the MNPs (Fl-DCCMNPs, Figure 1e, Figure S1a). Dynamic light scattering (DLS) analysis showed a slight increase in the hydrodynamic size of the MNPs after the serial conjugation of ProA (17 nm) and DCC Ab (21 nm) (Figure 1f). The size of the DCC-MNPs after incubation in culture media remained almost identical, without any signs of aggregation (Figure S1b). iN cells were directly derived from primary mouse embryonic fibroblasts (pMEFs) by the overexpression of three neuronal-specific transcription factors (Brn2, Ascl1, Myt1l).29−31 A considerable level of DCC expression was observed in the reprogrammed iN cells, which was ∼48% of pNeurons (Figure S2a,b). Homogenous labeling of the iN cells with the DCC-MNPs
eventually converges to affect cytoskeletal dynamics of maneuvering and growth cone navigation.20,21 Netrins, a family of canonical guidance cues, are a typical model for axonal outgrowth in the developing nervous system,22 as they interact with receptors such as deleted in colorectal cancer (DCC) or uncoordinated-5 (UNC5) to attract or repel neurons, respectively.23−26 It was previously reported that netrin triggers the homogeneous clustering of DCC receptors and activates downstream signaling pathways for actin rearrangement to facilitate axonal outgrowth in primary cortical neurons (pNeurons).25−28 Here, magnetic nanoparticles (MNPs) were designed to specifically bind the extracellular part of the DCC receptor (DCC-MNPs) for the induction of DCC clusterization upon exposure to a magnetic field, which will activate the downstream signaling cascade and induce neurite extension, including axonal outgrowth, to connect the grafted cells with host neurons (Figure 1a−c). We chose iN cells generated via direct reprogramming. Considering the therapeutic prospects of reprogramming technology to generate neurons from somatic cells, their B
DOI: 10.1021/acs.nanolett.9b02756 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters
Figure 2. Neurite outgrowth from iN cells artificially induced by DCC-MNPs under the uniform magnetic field (UMF). (a) Schematic illustration of the three-channel microfluidic device design and dimensions in a 2D top view and 3D side view. (b) Colored map of the strength of the uniform UMF inside chnmid of the microfluidic device (top). Magnetic field strength graph (bottom) (red lines, magnetic field strength along the x axis, Bx; blue lines, magnetic field strength along the y axis, By; dotted lines, simulation results along each axis; solid lines, linear fit for each magnetic field strength). (c) Immunostaining analysis of iN cells for Tau1 and Tuj1 (scale bar = 100 μm). (d, e) Quantification of the relative ratio of axons (Tau1, d) and neurites (Tuj1, e) between the left and right microgrooves. (f) Measurement of axon length in all groups (Tukey box and whisker plot; ◆, mean; **P < 0.01 versus the no treatment (NT) group, ++P < 0.01 versus the UMF only group, ##P < 0.01 versus the DCC-MNP with no UMF group).
was confirmed through detection of green fluorescence from the Fl-DCC-MNPs and red fluorescence after treatment with secondary antibody against the primary DCC Ab (Figure S2c). The mode of the magnetic field directed toward the biological sample can be modulated. A single magnet was used to generate a gradient magnetic field (GMF, >20 T/m, Figure S3) within the sample region, while two magnets were used to have a relatively uniform magnetic field (UMF,
