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Brain and Spinal Cord Injury Repair by Implantation of Human Neural Progenitor Cells Seeded Onto Polymer Scaffolds

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Brain and Spinal Cord Injury Repair by Implantation of Human Neural Progenitor Cells Seeded Onto Polymer Scaffolds

Jeong Eun Shin et al. Exp Mol Med.

Abstract

Hypoxic-ischemic (HI) brain injury and spinal cord injury (SCI) lead to extensive tissue loss and axonal degeneration. The combined application of the polymer scaffold and neural progenitor cells (NPCs) has been reported to enhance neural repair, protection and regeneration through multiple modes of action following neural injury. This study investigated the reparative ability and therapeutic potentials of biological bridges composed of human fetal brain-derived NPCs seeded upon poly(glycolic acid)-based scaffold implanted into the infarction cavity of a neonatal HI brain injury or the hemisection cavity in an adult SCI. Implantation of human NPC (hNPC)-scaffold complex reduced the lesion volume, induced survival, engraftment, and differentiation of grafted cells, increased neovascularization, inhibited glial scar formation, altered the microglial/macrophage response, promoted neurite outgrowth and axonal extension within the lesion site, and facilitated the connection of damaged neural circuits. Tract tracing demonstrated that hNPC-scaffold grafts appear to reform the connections between neurons and their targets in both cerebral hemispheres in HI brain injury and protect some injured corticospinal fibers in SCI. Finally, the hNPC-scaffold complex grafts significantly improved motosensory function and attenuated neuropathic pain over that of the controls. These findings suggest that, with further investigation, this optimized multidisciplinary approach of combining hNPCs with biomaterial scaffolds provides a more versatile treatment for brain injury and SCI.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
In vitro characterization of primary hNPCs seeded on a fibrous PGA scaffold. ad Seven days after being seeded, cells are able to attach, pervade, and grow exuberantly throughout the PGA fibers. The hNPCs predominantly differentiate into NF+ and TUJ1+ neurons (green) with neuronal processes that adhere to the PGA fibers (arrowheads in a, d). The PGA fibers are indicated by arrows in a. Many of the DAPI+ hNPCs (b, blue) are co-labeled with anti-NF antibody, seen under dual-filter microscopy in c. e, f The hNPCs differentiate not only into neurons but also into glial cells. Some human cells express GFAP, an astrocyte/immature cell marker (arrows in e), and a few cells express O4, an oligodendrocyte progenitor marker (arrows in f). The data shown are representative images. Scale bars: a 100 μm; d 10 μm
Fig. 2
Fig. 2
Transplantation of hNPC–PGA complexes into the infarction cavity of a HI brain injury. a Two weeks after transplantation (Tx) of the hNPC–PGA complexes (an arrow) into the infarction cavity indicated by asterisk, hNestin+ donor-derived cells (green) show robust engraftment within the injured area. The PGA fibers have begun to biodegrade (green strands, arrowheads). bg Some hNuc+ donor-derived cells (red in b, d, e, g) show co-localization with NF (blue arrows in b, c, d) with extended neuronal processes (white arrows in c, d; n = 15) or GFAP (white arrows in e, f, g; n = 15). The PGA fibers are indicated by arrowheads in bg. hk The de novo tissue following Tx of the hNPC–PGA complex into the infarction cavity (an asterisk in h) is connected to the host brain tissue by multiple NF+ neuronal processes (arrowheads in i, j, k) from host-derived and/or donor-derived neurons. Some donor-derived cells are identified by anti-hNuc staining (red arrows in h, I, j). The PGA fibers are indicated by green arrows in k. l, m The tract tracer BDA-FITC was injected into the contralateral intact cortex at 10 weeks following implantation of the hNPC–PGA complex into the infarction cavity. Axonal projections labeled green with fluorescein can be visualized from the cerebral cortex and external capsule (EC) of the intact hemisphere, projecting through the corpus callosum (CC) and toward the implantation site of the hNPC–PGA complex within the injured ipsilateral cortex and penumbra (l). Some BDA-FITC + anterograde-labeled processes from neurons in the contralateral intact hemisphere are observed sprouting their axons toward the implantation site of the hNPC–PGA complex (l). The BDA-FITC+ retrograde-labeled cell body and cellular processes of a neuron-like cell in the ipsilateral cortical penumbra indicated by an asterisk (in l) are well visualized at higher magnification (m). The data shown are representative images. Scale bars: a, l 500 μm; b, e, h, m 20 μm
Fig. 3
Fig. 3
Transplantation of hNPC–PGA complexes into the hemisection cavity of a SCI. a Spinal cord of an untransplanted rat subjected to complete transection of the left half of the spinal cord (arrow). b A piece of the hNPC–PGA complex was transplanted into the hemisection cavity of the spinal cord immediately after the induction of SCI. At 4 weeks post-transplantation, the hNPC–PGA complex appears to have filled the cavity (arrow) and become incorporated into the injured spinal cord. c At 2 weeks following Tx of a hNPC–PGA complex into the hemisection cavity, hNuc+ donor-derived cells (green) show robust engraftment within the injured area and adjacent intact spinal cord. d, e At 6 weeks following Tx of the hNPC–PGA complex, the de novo tissue within the hemisection cavity displays an intricate network of multiple long, branching NF+ (red) processes (arrowheads in d) and many very long, complex CGRP+ sensory neuronal processes (green; arrowheads in e) within the injury epicenter. The PGA fibers are indicated by arrows in e. f, g BDA tracing of the corticospinal fibers in a hNPC–PGA implanted animal. BDA+ fibers (green) are observed through the injury epicenter (arrowheads in f) and caudal to the injury in the same cord as in f (arrowheads in g). hr Coronal sections of a rat brain showing FB and/or DiI-labeled neuronal cell bodies in different areas of the brain 3 weeks after FB and DiI were injected into both left and right sides of the spinal cord caudal to the injury site in a hNPC–PGA implanted rat. While many FB+ (blue) and/or DiI+ (red) retrograde-labeled neurons (arrows) are identified in the ipsilateral (Ipsi) frontal cortex (FC; in i), deep subcortex (DS in jl), and midbrain (MBr in mo), relatively few FB-labeled and/or DiI-labeled neurons are found in the contralateral (Cont) FC (arrowheads in hh‴), DS (arrowheads in jl), and MBr (arrowheads in (pr). (h′h) The co-localization of retrograde tracers FB and DiI in the cell body and apical dendrite of a pyramidal neuron in the contralateral FC is easily visualized at high magnification (arrowheads). (jl) FB-labeled and/or DiI-labeled neurons are located in both the ipsilateral (arrows) and the contralateral DS (arrowheads) divided by the third ventricle (3rd V; midline delineated with white dotted lines). mr FB-labeled and/or DiI-labeled cells are found in both the ipsilateral (arrows in (mo) and the contralateral MBr (arrowheads in pr). The data shown are representative images. Scale bars: a 500 μm; c, d, e, h, h′, k 100 μm; g 10 μm
Fig. 4
Fig. 4
Implantation of a hNPC–PGA complex into HI brain injury inhibits glial scar formation and the gliotic response and increases neovascularization. a, b The hNPC–PGA Tx into the infarction cavity, indicated by an asterisk (in a), leads to a significant decrease in the density of GFAP staining (green) at the lesion epicenter, while there is intense GFAP staining in the peri-infarct area at 8 weeks post-transplantation (arrowheads in a, b). A few of hNuc+ donor-derived cells (red) are found within the newly formed tissue by implantation of the hNPC–PGA complex (arrows in b). f, i The PGA-alone Tx also causes a significant decrease in GFAP staining (red) in the injury epicenter (an arrow in f), while there is highly increased GFAP staining (red) in the injury epicenter and peri-infarct area of vehicle-injected (Vehicle Inj.) animal at 8 weeks post-injection (an arrow in i). c, g, j Tx of hNPC–PGA or PGA-alone into the infarction cavity causes less Iba1 staining (green) in the injury epicenter than vehicle injection (arrows in c, g, j, respectively). d, e, h, k At the same time post-transplantation, hNPC–PGA Tx causes highly significantly more CD31+ (red) small blood vessels in the injury epicenter (arrowheads in d, e) than implantation of PGA alone and injection of the vehicle (arrows in h, k). The data shown are representative images. Scale bars: a, d 500 μm; e, f, i 200 μm
Fig. 5
Fig. 5
Implantation of a hNPC–PGA complex into a SCI inhibits glial scar formation and the gliotic response and increases neovascularization. a, b Tx of hNPC–PGA into the hemisection cavity causes a significant decrease in the density of GFAP staining (green) at the lesion epicenter (white arrows in a; asterisk in b), while there is intense GFAP staining in the perilesion area at 6 weeks post-transplant (arrowheads in a, b). The PGA fibers are indicated by red arrows in a. e, h Tx of PGA alone also leads to significantly lower GFAP staining (green) in the injury epicenter (asterisk in e), while there is significantly more GFAP staining (green) in the injury epicenter and perilesion area of the vehicle-injected (Vehicle Inj.) animal at 6 weeks post-injection (arrowheads in h). c, f, i Tx of hNPC–PGA and PGA alone into the hemisection cavity causes less Iba1 staining (red) in the injury epicenter (asterisk in c, f), while there is highly increased Iba1 staining (red) in the injury epicenter and perilesion area of the vehicle-injected animal at 6 weeks post-injection (arrowheads in i). (d, g, j) At the same time post-transplantation, hNPC–PGA Tx causes highly significantly more vWF+ (red) small blood vessels (arrowheads in d) in the injury epicenter (asterisk in d) than implantation of PGA alone or injection of the vehicle (arrowheads in g, j). An asterisk (* in g, j) indicates the injury epicenter. The data shown are representative images. Scale bars: a 500 μm; g 200 μm
Fig. 6
Fig. 6
Behavioral performance of animals treated with hNPC–PGA, PGA alone, hNPCs, or vehicle after HI brain injury or SCI. a, b The results of the neurological test (a) and the latency to fall in the accelerating rotarod task (b) in HI brain injury are shown at 3, 5, 7, 9, and 11 weeks post-transplantation. a * and ** indicate a significant difference between the hNPC–PGA-treated and vehicle-treated groups, # exhibits a significant difference between hNPC-treated and vehicle-treated groups, and +++ represents a significant difference between sham controls and vehicle-injected group. b ** indicates a significant difference between the hNPC–PGA-treated and vehicle-treated groups, and + and +++ represent a significant difference between sham controls and vehicle-injected group. c, d The BBB open-field walking scores for the ipsilateral lesioned (c) and contralateral unlesioned hindlimbs (d) after SCI are shown at 1, 7, 14, 21, 28, 35, 42, 49, and 56 days post-transplantation. * and ** indicate a significant difference between the hNPC–PGA-treated and vehicle-treated groups in c, d. e Von Frey test for mechanical allodynia on ipsilateral lesioned hindlimbs after SCI are shown at a day before SCI, and 14, 28, 42, and 56 days post-transplantation. The data represent the mean ± SEM. (*, +P < 0.05 vs. vehicle; **P < 0.01 vs. vehicle; +++P < 0.001)

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