Transplantation of Human-Fetal-Spinal-Cord-Derived NPCs Primed with a Polyglutamate-Conjugated Rho/Rock Inhibitor in Acute Spinal Cord Injury

Abstract

Neural precursor cell (NPC) transplantation represents a promising therapy for treating spinal cord injuries (SCIs); however, despite successful results obtained in preclinical models, the clinical translation of this approach remains challenging due, in part, to the lack of consensus on an optimal cell source for human neuronal cells. Depending on the cell source, additional limitations to NPC-based therapies include high tumorigenic potential, alongside poor graft survival and engraftment into host spinal tissue. We previously demonstrated that NPCs derived from rat fetal spinal cords primed with a polyglutamate (PGA)-conjugated form of the Rho/Rock inhibitor fasudil (PGA-SS-FAS) displayed enhanced neuronal differentiation and graft survival when compared to non-primed NPCs. We now conducted a similar study of human-fetal-spinal-cord-derived NPCs (hfNPCs) from legal gestational interruptions at the late gestational stage, at 19-21.6 weeks. In vitro, expanded hfNPCs retained neural features, multipotency, and self-renewal, which supported the development of a cell banking strategy. Before transplantation, we established a simple procedure to prime hfNPCs by overnight incubation with PGA-SS-FAS (at 50 μM FAS equiv.), which improved neuronal differentiation and overcame neurite-like retraction after lysophosphatidic-acid-induced Rho/Rock activation. The transplantation of primed hfNPCs into immune-deficient mice (NU(NCr)-Foxn1^nu) immediately after the eighth thoracic segment compression prompted enhanced migration of grafted cells from the dorsal to the ventral spinal cord, increased preservation of GABAergic inhibitory Lbx1-expressing and glutamatergic excitatory Tlx3-expressing somatosensory interneurons, and elevated the numbers of preserved, c-Fos-expressing, activated neurons surrounding the injury epicenter, all in a low percentage. Overall, the priming procedure using PGA-SS-FAS could represent an alternative methodology to improve the capabilities of the hfNPC lines for a translational approach for acute SCI treatment.

Keywords: NPC transplantation; Rho/ROCK kinase inhibition; cell priming; human fetal neural precursor; spinal cord injury.

Figure 1

Cytoarchitecture of the human fetal spinal cord. (A) Transmission electron microscopy (TEM) images of the central canal of the human fetal spinal cord (scale bar (from left to right) = 20 µm, 10 µm, 2 µm, and 500 nm). The central canal is formed by ependymal cells organized as a pseudostratified epithelium with a large number of cilia in the apical zone (white arrow), nuclei with condensed chromatin (*), and large apical junction complexes (white arrowheads). (B) Representative coronal immunostaining images (complete coronal sections in upper panels and magnified view of the corresponding CC area in the lower panels) of human fetal spinal cords for SOX2 (yellow), Ki67 (yellow), Nestin (green), Gfap (red), NeuN (red), and Olig2 (yellow) (white bar scale= 500 µm; red bar scale = 50 µm).

Figure 2

hfNPCs display proliferative potential and express canonical NPC markers in vitro. (A) Phase contrast image from hfNPCs as neurospheres-like cultures forming primary (black arrows) and secondary (white arrows) neurospheres as a hallmark of self-renewal in the presence of mitogens at passage 1. Scale bar = 200 µm. (B) Representative phase contrast image of hfNPC culture in adherent conditions in laminin-coated wells when cultured at passages 2–5 (Scale bar = 20 µm). (C) Doubling time (t) analysis of hfNPCs (black line; data presented as the mean ± SEM (n = 5 samples)) and one rat fetal NPC sample (red line) growing in adherent conditions over five passages. (D) The pinwheel cytoarchitecture (indicated with a white dotted line) of hfNPC at passage 1 from neurosphere-like cultures is highlighted after β-catenin (red; delimiting the cell perimeter) and γ-tubulin (green; for cilia detection) staining (scale bar = 20 µm (left panel) and 10 µm (right panel)). (E) Representative images from nuclear staining of DAPI (blue), Ki67 (red), and BrdU (green) of hfNPCs in adherent conditions (scale bar = 10 µm). Right panel: Quantification of the percentage of positive cells for BrdU and Ki67. Data presented as the mean ± SEM (n = 3 samples). (F) Left panel: Representative images of immunofluorescent staining for Notch, Pax6, Nestin, Sox2, FoxJ1, DCX, and neurogenin (scale bar = 20 µm). Right panel: Quantification of the percentage of positive cells for each canonical neural progenitor cell marker. Data presented as the mean ± SEM (n = 5 samples).

Figure 3

PGA-SS-FAS priming process enhances neuronal and oligodendrocyte differentiation and induces neurite outgrowth in a model of neurite retraction. (A) Representative immunofluorescence images of hfNPCs using β-III-tubulin for immature neurons (green), GFAP for astrocytes (red), Olig2 for oligodendrocytes (green), NeuN for mature neurons (red), and DAPI (blue) (scale bar = 20 µm) (positive nuclear staining is indicated with an arrow) in the presence of the PGA-SS-FAS compound for inducing the priming process (PGA-SS-FAS) or its vehicle (control). (B) Quantification of the percentage of positive cells for each of the indicated cell markers with (black bars) or without (white bars) 24 h PGA-SS-FAS priming. Data presented as the mean ± SEM determined by Student’s unpaired t-test (n = 3). ** p < 0.01, *** p < 0.001 vs. hfNPCs. (C) Representative immunofluorescence images of β-III-tubulin (green) and DAPI (blue) staining during the in vitro neurite retraction assay induced by lysophosphatidic acid (LPA) treatment during 24 h in hfNPC adherent cultures. Yellow arrows indicate neurite-like processes emanating from neuronal progenitors in the control condition (in the absence of LPA) and in co-treated cultures (LPA + PGA-SS-FAS); red arrows indicate rounded cells 24 h after LPA treatment and induced neurite-like retraction (scale bar = 50 µm). (D) Neurite length quantification presented as the mean ± SEM determined by one-way ANOVA with the Tukey multiple comparison test (n = 3). ** p < 0.01, *** p < 0.001 vs. control; +++ p < 0.001 vs. control.

Figure 4

Quantification, distribution, and phenotypic characterization of non-primed and primed hfNPCs post-transplantation. (A) Representative images of GFP staining for hfNPC-grafted cells in spinal cord horizontal sections for the indicated groups (green) (scale bar = 50 µm). (B) Quantification of GFP-positive transplanted cells normalized to the total measured thickness in µm². (C) quantification of GFP-positive transplanted cells in absolute numbers every 5th 20 mm section. (D) Quantification of the dorso-ventral distribution of the GFP-positive cells expressed in percentage of the total number of quantified cells. (E) representative images of c-Fos+ (red) and GFP (green) and the merged image including DAPI (blue) -positive immunostaining of grafted GFP-positive hfNPCs. (F) Quantification of the percentage of c-Fos-positive hfNPCs in the primed or non-primed groups, presented as the mean ± SEM and determined by Student’s unpaired t-test (n = 3). (* p < 0.05). (G) Representative images of immunofluorescent staining for GFP- (green), Lbx1- (red), and Tlx3- (yellow) positive grafted cells and merged images with DAPI (blue) of both experimental groups (scale bar = 25 µm). (H) Quantification of the percentage of Lbx1. (I) Tlx3-positive hfNPCs presented as the mean ± SEM and determined by Student’s unpaired t-test (n = 3)). (J) Representative immunostainings of c-Fos (yellow), NeuN (red), and DAPI (blue) (white scale bar = 500 µm; red scale bar = 25 µm). (K) Quantification of NeuN-positive cells in the rostral, lesion normalized to the total measured thickness in µm², and caudal sites. (L) Quantification of NeuN-positive cells in the lesion site; data presented as the mean ± SEM determined by one-way ANOVA with Tukey’s multiple comparison test (n = 3). (M) Quantification of NeuN/c-Fos-double-positive cells in the lesion site normalized to the total measured thickness in µm². Data presented as the mean ± SEM determined by one-way ANOVA with Tukey’s multiple comparison test (n = 3). (N) Representative immunostainings of (top panel) Lbx1 (red), GFP (green), and DAPI (blue) and (bottom panel) Tlx3 (red), GFP (green), and DAPI (blue) (scale bar = 50 µm) for the indicated experimental groups. (O) Quantification of Lbx1. (P) Tlx3-positive cells in the area surrounding the graft and lesion normalized to the total measured thickness in µm². Data presented as the mean ± SEM and determined by one-way ANOVA with Tukey’s multiple comparison test (n = 3) (* p < 0.05, *** p < 0.001 (* p < 0.05, *** p < 0.001, ns = non-significant).