Enhancing neural stem cell integration in the injured spinal cord through targeted PTEN modulation

Abstract

JOURNAL/nrgr/04.03/01300535-202604000-00039/figure1/v/2025-06-30T060627Z/r/image-tiff Spinal cord injury results in permanent loss of neurological functions due to severance of neural networks. Transplantation of neural stem cells holds promise to repair disrupted connections. Yet, ensuring the survival and integration of neural stem cells into the host neural circuit remains a formidable challenge. Here, we investigated whether modifying the intrinsic properties of neural stem cells could enhance their integration post-transplantation. We focused on phosphatase and tensin homolog (PTEN), a well-characterized tumor suppressor known to critically regulate neuronal survival and axonal regeneration. By deleting Pten in mouse neural stem cells, we observed increased neurite outgrowth and enhanced resistance to neurotoxic environments in culture. Upon transplantation into injured spinal cords, Pten-deficient neural stem cells exhibited higher survival and more extensive rostrocaudal distribution. To examine the potential influence of partial PTEN suppression, rat neural stem cells were treated with short hairpin RNA targeting PTEN, and the PTEN knockdown resulted in significant improvements in neurite growth, survival, and neurosphere motility in vitro . Transplantation of shPTEN-treated neural stem cells into the injured spinal cord also led to an increase in graft survival and migration to an extent similar to that of complete deletion. Moreover, PTEN suppression facilitated neurite elongation from NSC-derived neurons migrating from the lesion epicenter. These findings suggest that modifying intrinsic signaling pathways, such as PTEN, within neural stem cells could bolster their therapeutic efficacy, offering potential avenues for future regenerative strategies for spinal cord injury.

Keywords: PTEN; graft axon growth; graft survival; neural stem cell; regeneration; spinal cord injury; transplantation.

Figure 1

Influence of Pten gene deletion on mouse NSCs in vitro. (A) Verification of PTEN deletion and enhanced phosphorylation of S6 ribosomal protein. Cultured mouse spinal cord NSCs from Pten^fl/fl mouse were transfected with either AAV2-GFP (GFP) or AAV2-Cre-GFP (CRE). One week after transduction, neurospheres were collected and subjected to western blot analysis. Beta-actin was probed as a loading control. (B) Representative fluorescence images of GFP-positive neurospheres (green). Scale bars: 100 μm. (C) Quantitative graphs comparing the number and size of neurospheres. Scale bars: 50 μm. (D) Representative images of NSC-derived neurons grown on either growth-permissive (laminin) or growth-inhibitory (CSPG) substrate. Neurites were visualized by immunocytochemistry using anti-βIII tubulin (magenta) antibody. Scale bars: 50 μm. (E) Quantitative graphs comparing the neurite length of NSC-derived neurons grown on either growth-permissive (laminin) or growth-inhibitory (CSPG) substrate. (F, G) Comparison of NSC survival measured by MTT assay (F) and extent of NSC death measured by far red fluorescence-based cell sorting (G). Control (GFP) or Pten-deleted (CRE) mouse NSCs were treated with NaAsO₂ to mimic a degeneration-prone injury environment. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test for C and E; two-way analysis of variance followed by post hoc Bonferroni’s multiple comparison test for F and G). Each data point represents an independent culture. CSPG: chondroitin sulfate proteoglycan; GFP: green fluorescent protein; NaAsO₂: sodium arsenite; NSC: neural stem cells; PTEN: phosphatase and tensin homolog.

Figure 2

Pten deletion enhances the survival and migration of grafted NSCs in vivo. (A) Longitudinal spinal cord section from NOD/SCID immune-deficient mice transplanted with either mouse spinal cord NSCs from Pten^fl/fl mouse transfected with either AAV2-GFP (GFP) or AAV2-Cre-GFP (CRE) NSCs (green). Fluorescence signals were augmented by immunohistochemical staining using an antibody against GFP. Animals were sacrificed at 8 weeks after transplantation. Dotted rectangles in low-magnification images were magnified in the separate inset images below. Arrows indicate elongated cytoplasmic processes from Pten-deleted located far from the injection site. Scale bars: 200 μm. (B–D) Quantitative graphs comparing the number of surviving NSCs (B), areas occupied by GFP-positive grafts (C), and the longest distance of NSCs from the injection site in a rostrocaudal direction (D) between control (GFP) and Pten-deleted (CRE) NCSs. *P < 0.05, **P < 0.01 (unpaired Student’s t-test). Each data point in all the graphs represents a single animal. GFP: Green fluorescent protein; NSC: neural stem cells.

Figure 3

Differentiation of NSCs with Pten deletion in injured spinal cord. (A, B) Representative images of the spinal cord sections where immunohistochemical staining of βIII-tubulin (Tuj1, Magenta) (A) and glial fibrillary acidic protein (GFAP, magenta) (B) was performed. Scale bars: 20 μm. (C, D) Quantitative graphs of the percent GFP positive NSCs (green) colocalized with Tuj1 (C) and GFAP (D). n = 6 for each group. Each data point in all the graphs represents a single animal. DAPI: 4′,6-Diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; NSC: neural stem cells.

Figure 4

Influence of shRNA-mediated Pten silencing on rat NSCs in vitro. (A) Verification of a partial reduction of PTEN expression and an increase in phosphorylation of S6 ribosomal protein. Cultured rat spinal cord NSCs (Green) were transfected with either AAV2-GFP (GFP) or AAV2-shRNA targeting Pten gene (shPTEN). One week after transduction, neurospheres were collected and subjected to western blot analysis. Beta-actin was probed as a loading control. (B) Representative fluorescence images of GFP-positive neurospheres (green). Scale bars: 100 μm. (C) Quantitative graphs comparing the number and size of neurospheres. (D) Representative images of NSC-derived neurons grown on either growth-permissive (laminin) or growth-inhibitory (CSPG) substrate. Neurites were visualized by immunocytochemistry using anti-βIII tubulin (Magenta) antibody. Scale bars: 50 μm. (E) Quantitative graphs comparing the neurite length of NSC-derived neurons grown on either growth-permissive (laminin) or growth-inhibitory (CSPG) substrate. (F, G) Comparison of NSC survival measured by MTT assay (F) and extent of NSC death measured by far red fluorescence-based cell sorting (G). Control NSCs (GFP) or NSCs with Pten knockdown (shPTEN) rat NSCs were treated with sodium arsenite (NaAsO₂) to mimic a degeneration-prone injury environment. NS indicates not significant. (H) Representative snapshot images from the movie clips recorded during the neurosphere motility assay. Neurospheres derived from Control NSCs (GFP) or NSCs with Pten knockdown (shPTEN) rat NSCs were seeded on a 24-well culture plate and time-lapse images were obtained for 48 hours. Each snapshot was taken at the time point marked above. Scale bars: 200 μm. (I) Quantitative graphs comparing the total distance and the velocity of motile neurospheres from each group. Each data point indicates an average of at least four neurospheres from one live imaging session. Data from three live imaging sessions per group were included. Each data point represents an independent culture. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test for C, E, I; two-way analysis of variance followed by post hoc Bonferroni’s multiple comparison test for F and G). CSPG: Chondroitin sulfate proteoglycan; GFP: green fluorescent protein; NSC: neural stem cells.

Figure 5

Influence of shRNA-mediated Pten silencing on the survival of NSC grafts in vivo. (A, B) Representative images of the longitudinal spinal cord sections from rats transplanted with either control NSCs (GFP, Green) or NSCs with Pten knockdown (shPTEN, green). Animals were sacrificed at 2 (A) and 8 weeks (B) after transplantation. Dotted rectangles in low-magnification images were magnified in the separate inset images below. Scale bars: 200 μm. (C, D) Quantitative graphs of the success rate at 2 and 8 weeks following transplantation. n = 19 (GFP = 9, shPTEN = 10) for the 2-week survival and 19 (GFP = 11, shPTEN = 8) for the 8-week survival experiments. (E, F) Quantitative graphs to compare the number of surviving GFP-positive NSCs and the length of cellular processes between NSCs in GFP and shPTEN groups at 2 (E) and 8 weeks (F). The data from animals determined as failure were not included in these analyses. *P < 0.05, **P < 0.01 (unpaired Student’s t-test). (G) Representative images of the serotonin (5-HT, red) axons from the brainstem growing into the lesion where NSC grafts (green) were present. Dotted rectangular regions were magnified with orthographic projections on the right side. White arrows indicate 5-HT axonal boutons colocalized with GFP-positive NSCs. Scale bars: 10 m. Each data point in all the graphs represents a single animal. 5-HT: 5-Hydroxytriptamine; GFP: green fluorescent protein; NSC: neural stem cells.

Figure 6

Pten silencing leads to extensive migration of NSCs and elongation of neurites from NSC-derived neurons in vivo. (A, B) Representative images of the longitudinal spinal cord sections showing rostrocaudal migration of grafted NSCs (green) from the epicenter at 2 (A) and 8 weeks (B) after transplantation. Compared to control NSCs (GFP), NSCs with Pten knockdown (shPTEN) exhibited extensive migration, especially at 8 weeks following transplantation. Dotted rectangles in low-magnification images were magnified in the separate inset images below. Scale bars: 200 μm. White arrows indicate the elongated morphology of NSCs at the leading edge of the migration. (C, D) Quantitative graphs comparing the migration distance between control NSCs (GFP) or NSCs with Pten knockdown (shPTEN) at 2 (C) and 8 weeks (D). Only the data from the animals with graft success were included. (E) Immunohistochemical localization of neurofilament positive axons (magenta) growing from grafted NSCs (green) in the spinal cord at 8 weeks after transplantation. Antibodies against medium-chain NFM were used as a marker of axons. White arrows indicate GFP-positive graft-derived processes colocalized with NFM. Scale bars: 50 μm. (F) A quantitative graph of the length of NFM-positive elongated progresses growing from GFP-positive grafts. n = 5 and 6 for control GFP and shPTEN groups. *P < 0.05, **P < 0.01 (unpaired Student’s t-test). Each data point in all the graphs represents a single animal. GFP: Green fluorescent protein; NFM: neurofilament M; NSC: neural stem cells.