An NT-3-releasing bioscaffold supports the formation of TrkC-modified neural stem cell-derived neural network tissue with efficacy in repairing spinal cord injury

Abstract

The mechanism underlying neurogenesis during embryonic spinal cord development involves a specific ligand/receptor interaction, which may be help guide neuroengineering to boost stem cell-based neural regeneration for the structural and functional repair of spinal cord injury. Herein, we hypothesized that supplying spinal cord defects with an exogenous neural network in the NT-3/fibroin-coated gelatin sponge (NF-GS) scaffold might improve tissue repair efficacy. To test this, we engineered tropomyosin receptor kinase C (TrkC)-modified neural stem cell (NSC)-derived neural network tissue with robust viability within an NF-GS scaffold. When NSCs were genetically modified to overexpress TrkC, the NT-3 receptor, a functional neuronal population dominated the neural network tissue. The pro-regenerative niche allowed the long-term survival and phenotypic maintenance of the donor neural network tissue for up to 8 weeks in the injured spinal cord. Additionally, host nerve fibers regenerated into the graft, making synaptic connections with the donor neurons. Accordingly, motor function recovery was significantly improved in rats with spinal cord injury (SCI) that received TrkC-modified NSC-derived neural network tissue transplantation. Together, the results suggested that transplantation of the neural network tissue formed in the 3D bioactive scaffold may represent a valuable approach to study and develop therapies for SCI.

Keywords: Neural network tissue; Neurotrophin-3; Self-organization; Spinal cord injury; Tropomyosin kinase receptor C.

Graphical abstract

Fig. 1

Construction of the neurotrophin-3 (NT-3)/fibroin-releasing gelatin sponge (NF-GS) scaffold, and NT-3 enhances the survival of neural stem cells (NSCs) seeded in the scaffold. (A) Immunoelectron micrograph showing that NT-3 (probed using round, electron-dense nanogold particles; pseudocolored in green) was deposited on the surface of the gelatin sponge. The inset is a scanning electron photomicrograph showing an overview of the NF-GS scaffold. (B) Higher magnification image from (A) showing the nanogold particle-enriched area. (C) An overview of the NF-GS scaffold (3 mm in diameter and 2 mm in length). (D) Cumulative release of NT-3 from one NF-GS scaffold over 28 days in vitro. (E) Tensile stress–strain curves for poly(lactic-co-glycolic acid) (PLGA) from three samples (1, 2 and 3). (F) Tensile stress–strain curves for the GS scaffold from three samples (1, 2 and 3). (G) Tensile stress–strain curves for the NF-GS scaffold from three samples (1, 2 and 3). (H) A Cell Counting Kit-8 (CCK8) assay showed that cells seeded in the NF-GS scaffold, fibroin GS scaffold (F-GS), or GS scaffold exhibited similar viability. The GS + NSCs group was used as the benchmark (100%). (I)–(L) The rate of cell apoptosis was lower in the NF-GS scaffold than in the other two scaffolds. (M)–(U) After 14 days of culture, NSCs (green) differentiated into microtubule-associated protein 2 (Map2)-positive neurons, glial fibrillary acidic protein (GFAP)-positive astrocytes, and myelin basic protein (MBP)-positive oligodendrocytes in the NF-GS, F-GS and GS scaffolds. (V) Bar chart showing that there were fewer GFAP-positive cells in the NF-GS group relative to the other two groups; *P < 0.05. Hoe: Hoechst33342. Scale bars: 5 μm in (A), 0.5 μm in (B), 20 μm in (I)–(K) and 25 μm in (M)–(U). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

The spatial and temporal patterns of neurotrophin-3 (Ntf3) and neurotrophic tyrosine kinase, receptor, type 3 (Nrtk3) expression during neurogenesis in the mouse embryonic spinal cord. (A) Uniform manifold approximation and projection (UMAP) plot showing all the cells of the single cell atlas. Cells are colored by their cell-type annotation and numbered according to the legend (right): 0, mesenchyme I; 1, neural progenitor I; 2, neural progenitor II; 3, neural crest; 4, neuron dl5–6 II; 5, neuron dl5–6 I; 6, neuron V0-1-V2b; 7, mesenchyme II; 8, neural progenitor III; 9, neural progenitor V; 10, neuron dl4 III; 11, neuron dl1-3-V3; 12, neuron dl4 II; 13, neural progenitor IV; 14, neural crest neurons; 15, motor neurons; 16, neuron dl4 I; 17, null neuron; 18, erythrocyte; 19, neuron V2a; 20, blood; 21, hematopoietic; and 22, myoblast. (B) UMAP plot showing the expression level of marker genes (Sox2, neural progenitors; Sox10, neural crest cells; Tubb3, neurons; Lmx1b, dl5–6 neurons; and Mnx1, motor neurons). The marked numbers are as in panel (A). (C) Bubble charts showing the temporal expression of Ntf3 and Ntrk3 in different dorsal–ventral (DV) domains during neurogenesis. The size of the circles indicates the mean expression of genes per stage and domain, and the color indicates the age of the sample. (D) Principal component analysis (PCA) projection of all neural cells, shown on a hexagonal heatmap along the pseudotime axis of neurogenesis. The hexagonal heatmaps indicate the expression pattern of Ntrk3, Ntf3, the pan-neural progenitor marker Sox2, and the neuronal marker, Tubb3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

The NF-GS scaffold promoted neuronal differentiation and the survival of seeded tropomyosin receptor kinase C (TrkC)-modified neural stem cells (NSCs) (TrkC-NSCs). (A)–(I) Green fluorescent protein (GFP)-TrkC-positive NSCs differentiated into microtubule-associated protein 2 (Map2-positive neurons, glial fibrillary acidic protein (GFAP)-positive astrocytes, and myelin basic protein (MBP)-positive oligodendrocytes in the NF-GS, F-GS, and GS scaffolds. (J) Bar chart showing that TrkC-NSCs differentiated more frequently into Map2-positive neurons and less frequently into GFAP-positive astrocytes in the NF-GS scaffold relative to the F-GS and GS scaffolds. (K) Schematic diagram illustrating the timepoints at which action potentials (APs) or excitatory postsynaptic currents (EPSCs) were recorded from TrkC-NSC-derived neurons. (L) The patch clamp recording method. (M) APs recorded in a TrkC-NSC-derived neuron in the NF-GS scaffold. (N)–(Q) Cell apoptosis among TrkC-overexpressing cells was lower in the NF-GS group than in the F-GS or GS groups; *P < 0.05. Hoe: Hoechst33342. Scale bars: 25 μm in (A)–(I) and 20 μm in (N)–(P). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

The NF-GS scaffold supported the formation of a neural network by seeded TrkC-modified NSCs (TrkC-NSCs). (A) TrkC-NSCs expressed both postsynaptic density protein 95 (PSD95, red), a postsynaptic marker, and the presynaptic marker synaptophysin (SYP, white) in the NF-GS scaffold after 14 days of culture. (B) A bar chart showing that the relative fluorescence intensity of both PSD95 and SYP was significantly higher in the NF-GS+TrkC-NSCs group than in the GS+NSCs group. (C)–(F) TEM revealed the presence of abundant intercellular junctions between TrkC-NSC-derived cells (Nuc: nucleus, blue pseudocolor; Mit: mitochondria). Some intercellular junctions resembled synapse-like features. (G)–(J) TrkC-NSC-derived neurons expressed the cholinergic marker choline acetyltransferase (G, inset), the glutamatergic marker glutamate transporter 1 (VGluT1) (H, inset), or the GABAergic marker gamma-aminobutyric acid (GABA) (I, inset). (K) Representative excitatory postsynaptic currents (EPSCs) recorded in TrkC-NSC-derived neurons cultured in the NF-GS scaffolds for 23 days; *P < 0.05. Hoe: Hoechst33342. Scale bars: 40 μm in (A), 1 μm in (C), 200 nm in (D)–(F) and 20 μm in (G)–(I). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

The formation of TrkC-modified NSC-derived neural tissue in the NF-GS scaffold. (A) Transmission electron photomicrographs showing neuropil-like structures represented by synaptically-dense regions in the neural tissue (Nuc: nucleus, blue pseudocolor). (B)–(D) A large number of synapse-like structures (arrows) were apparent in the neural tissue. (E)–(F) A typical myelin sheath seen in the neural tissue. (G) Scanning electron micrograph showing that cells (arrows) attached to the surface of the scaffold, with abundant processes (arrowheads) contacting each other. (H)–(L) TrkC-NSC-derived cells expressing integrin β1 attached to the laminin matrix in the NF-GS scaffold. Hoe: Hoechst33342. Scale bars: 2 μm in (A), 200 nm in (B)–(D), 20 μm in (E), 800 nm in (F), 300 nm in (G) and 40 μm in (H)–(L). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Neural network tissue (NNT) derived from TrkC-modified NSCs survived in the injury/graft site of the spinal cord for 8 weeks after transplantation. (A)–(C) Surviving GFP-positive donor cells were significantly more frequently observed in the NNT group than in the NSC group (without neural network induction). (D) and (E) Western blot analysis showing the differential GFP expression between the NNT group and the NSC group. (F) A bar chart showing the maintenance of the dominant neuronal population within the NNT graft composed of donor and endogenous Map2-immunopositive neurons. (G) and (H) Representative Map2-positive donor neurons (G) and GFAP-positive donor astrocytes. (I) Transmission electron micrograph showing a donor oligodendrocyte (nanogold particle labeling, arrows; Nuc: nucleus, blue pseudocolor) forming a myelin sheath to enwrap a host nerve fiber (asterisk). (J) GFP-positive donor neurons expressing synaptophysin (SYP), a presynaptic marker, and postsynaptic density protein95 (PSD95), a postsynaptic marker. (K) Immunoelectron micrograph showing that GFP- (DAB-labeled) and TrkC- (nanogold particle-labeled) positive donor neurons (Nuc: nucleus, blue pseudocolor) formed synaptic connections (green showing presynaptic element composed of axon terminal of a neuron), with identifiable presynaptic vesicles (yellow pseudocolor) and postsynaptic densities (arrows). The inset shows a higher magnification of the synaptic structure (arrows showing synaptic cleft). (L)–(O) Significant laminin deposition was observed in the injury/graft site and the rostral and caudal areas adjacent to the injury/graft site after NNT transplantation. Some donor cells expressed integrin β1. (P) Immunoelectron micrograph showing a GFP-positive donor cell (DAB-labeled) that has adhered to the linear laminin matrix (nanogold particle-labeled, arrows); *P < 0.05. Hoe: Hoechst33342. Scale bars: 1 mm in (A), (B) and (L); 20 μm in (G), (H), (J), (M), (N) and (O); 1 μm in (I) and (P); and 0.5 μm in (K). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

The donor neural network integrated with the descending and ascending neural circuits of the host. (A)–(C) Descending 5-HT-positive nerve fibers regenerated into the area rostral to the injury/graft site (B) and into the center of the injury/graft site (C), making contacts with GFP-positive donor cells within the neural network graft. (D) and (E) An immunoelectron micrograph showing that a 5-HT-positive (nanogold particle-labeled, arrowheads) nerve fiber formed contacts (arrows) with a neuron (Nuc: nucleus, blue pseudocolor) from the injury/grate site. (F)–(K) CGRP-positive nerve fibers regenerated into areas rostral (G) and caudal (H) to the injury/graft site, and some made close contacts with GFP-labeled donor cells within the neural network graft (I–K). (L) A PSD95-positive donor neuron contacting a CGRP-positive nerve fiber (arrows). (M) and (N) Immunoelectron micrograph showing the formation of synapses between a CGRP-positive nerve fiber (DAB-labeled) and an endogenous cell in the injury/graft site. (O) Immunoelectron micrograph showing a myelin sheath enwrapping a regenerated CGRP-positive fiber in the injury/graft site. (P)–(R) Immunoelectron micrograph showing the formation of synapse-like structures between two CGRP-positive nerve fibers (DAB-labeled) and a GFP-positive donor cell (nanogold particle-labeled). Hoe: Hoechst33342. Scale bars: 1 mm in (A) and (F); 20 μm in (B), (C) and (G)–(L); 0.5 μm in (D), (M), (O) and (P); 200 nm in (E), (N), (Q) and (R). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Grafted neural network tissue (NNT) improved motor function recovery and electrophysiological activity. (A) Frequent plantar placement with body weight support was observed only in the NNT group in the inclined grid test. (B) Basso–Beattie–Bresnahan (BBB) scoring showing that there was a significant improvement in hindlimb motor function after NNT transplantation. (C) Representative recorded cortical motor evoked potentials (CMEPs). (D) and (E) Rats in the NNT group had larger CMEP amplitudes and shorter CMEP latencies compared with those of rats in the control groups. *P < 0.05, vs. the spinal cord injury group; ^#P < 0.05, vs. the NF-GS group; ^&P < 0.05, vs. the NSCs group.