IL-10 lentivirus-laden hydrogel tubes increase spinal progenitor survival and neuronal differentiation after spinal cord injury

Abstract

A complex cellular cascade characterizes the pathophysiological response following spinal cord injury (SCI) limiting regeneration. Biomaterial and stem cell combination therapies together have shown synergistic effects, compared to the independent benefits of each intervention, and represent a promising approach towards regaining function after injury. In this study, we combine our polyethylene glycol (PEG) cell delivery platform with lentiviral-mediated overexpression of the anti-inflammatory cytokine interleukin (IL)-10 to improve mouse embryonic Day 14 (E14) spinal progenitor transplant survival. Immediately following injury in a mouse SCI hemisection model, five PEG tubes were implanted followed by direct injection into the tubes of lentivirus encoding for IL-10. Two weeks after tube implantation, mouse E14 spinal progenitors were injected directly into the integrated tubes, which served as a soft substrate for cell transplantation. Together, the tubes with the IL-10 encoding lentivirus improved E14 spinal progenitor survival, assessed at 2 weeks posttransplantation (4 weeks postinjury). On average, 8.1% of E14 spinal progenitors survived in mice receiving IL-10 lentivirus-laden tubes compared with 0.7% in mice receiving transplants without tubes, an 11.5-fold difference. Surviving E14 spinal progenitors gave rise to neurons when injected into tubes. Axon elongation and remyelination were observed, in addition to a significant increase in functional recovery in mice receiving IL-10 lentivirus-laden tubes with E14 spinal progenitor delivery compared to the injury only control by 4 weeks postinjury. All other conditions did not exhibit increased stepping until 8 or 12 weeks postinjury. This system affords increased control over the transplantation microenvironment, offering the potential to improve stem cell-mediated tissue regeneration.

Keywords: biomaterials; gene delivery; neural stem cells; spinal cord injury; tissue engineering.

Figure 1:

FLuc loaded PEG tubes sustained expression over a course of 12 weeks. Qualitatively, (A) bioluminescence was detected in highest concentrations at the injured spinal cord. (B) Bioluminescent signal of lentivirus-laden hydrogel tubes was assessed over a course of 12 weeks with higher expression compared to background across all times evaluated. n=6 per condition, * p<0.05, ** p<0.01, **** p<0.001.

Figure 2:

Macrophage infiltration is not exacerbated by tube implantation or E14 spinal progenitor transplantation. Macrophage densities in (A) SCI only, (B) Tubes only, (C)Tubes+IL-10, (D) E14 spinal progenitors only, (E) Tubes+E14, and (F) Tubes+E14+IL-10 in transverse tissue cross sections exhibit both M1 macrophages (F4/80⁺, red) denoted by ^ and M2 macrophages (F4/80⁺arginase⁺, red and green) denoted by _*. At 4 weeks after implantation, there were no differences in (G) total macrophage density, (H) M2 macrophage density, and (I) M2 percent across all conditions. Data are presented as mean ± SEM. n=4 per condition, 50 μm scale bars.

Figure 3:

(A) Experimental timeline detailed with major interventions. Delayed EGFP⁺ spinal progenitor transplantation into IL-10 encoding lentivirus loaded tubes 2 weeks post-injury improves survival. Five hydrogel tubes, either blank or IL-10 lentivirus loaded, were implanted directly after lateral C5 hemisection injury, and EGFP⁺ spinal progenitors were transplanted 2 weeks post-injury into (B) SCI only, (C) blank tubes, or (D) IL-10 lentivirus loaded tubes. Quantitatively, survival was assessed 4 weeks post-injury (2 weeks post-transplantation), and IL-10 lentivirus loaded tubes exhibited an increase in (E) cell density and (F) percent survival over the other two conditions. Data are presented as mean ± SEM. n=4 per condition, * p<0.05, ** p<0.01, 50 μm scale bars.

Figure 4:

Neuron formation at 12 weeks post-injury is increased when hydrogel tubes are implanted. (A) Formed neurons were observed at 12 weeks post-injury arising from both exogenous (EGFP⁺NeuN⁺|green⁺gray⁺, green arrow) and endogenous (BrdU⁺NeuN⁺|red⁺gray⁺, pink arrow) sources. (B) No statistically significant differences was observed in exogenous-sourced neuron density. (C) A significant increase in NeuN⁺ cells as a percent of total EGFP⁺ cells was observed when E14 spinal progenitors were transplanted into either blank tubes or IL-10 lentivirus-loaded tubes. (D) There were no statistically significant differences observed in endogenous-sourced neuron formation. Data are represented as mean ± SEM, n = 4–7 animals per conditions, * p<0.05, ** p<0.01, 50 μm scale bars.

Figure 5:

E14 spinal progenitor transplants into IL-10 lentivirus-laden tubes improve new axon formation at 12 weeks post-injury. Axon expression (NF-200⁺) in (A) SCI only, (B) Tubes only, (C) Tubes+IL-10, (D) E14 spinal progenitors only, (E) Tubes+E14, and (F) Tubes+E14+IL-10. Examples of (G) bundles and (H) elongated axons observed in the ipsilateral tissue. Quantitatively, animals receiving Tubes+E14+IL-10 exhibited a higher elongated axon density (I) in the ipsilateral tissue. Data are presented as mean ± SEM. n=6 animals per condition, * p<0.05, ** p<0.01, *** p<0.005, 200 μm scale bars (A-F), 50 μm scale bars (G-H).

Figure 6:

E14 spinal progenitor delivery increases axon remyelination 12 weeks post-injury. Axon (NF-200⁺, red) co-localization with myelin (MBP⁺, green) was assessed in (A) SCI only, (B) Tubes only, (C) Tubes+IL-10, (D) E14 spinal progenitors only, (E) Tubes+E14, and (F) Tubes+E14+IL-10. (G) E14 only transplants and Tubes+E14+IL-10 exhibited an increase in overall myelinated axon density compared to the SCI only condition. (H) As a percent, myelination was significantly increased in the E14 only condition compared to all other conditions. Data are represented as mean ± SEM, n = 4–7 animals per condition, * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001, 20 μm scale bars.

Figure 7:

Animals receiving E14 spinal progenitors transplanted into IL-10 lentivirus-laden tubes exhibited a quicker return of forelimb motor function. To assess mobility, a horizontal ladder beam test was used. Mice were trained on walking across the ladder prior to injury. Mobility was assessed through successful placements as a score out of 50 possible rungs. By 4 weeks post-injury Tubes+E14+IL-10 (a, p<0.005) had improved stepping compared to SCI only. At 8 weeks all conditions with tubes had improved stepping compared to (Tubes+E14, b, p<0.005; Tubes+IL-10, c, p<0.05; Tubes, d, p<0.05). At 12 weeks all conditions exhibited improved stepping (E14 only, e, p<0.01).