Defining recovery neurobiology of injured spinal cord by synthetic matrix-assisted hMSC implantation

Abstract

Mesenchymal stromal stem cells (MSCs) isolated from adult tissues offer tangible potential for regenerative medicine, given their feasibility for autologous transplantation. MSC research shows encouraging results in experimental stroke, amyotrophic lateral sclerosis, and neurotrauma models. However, further translational progress has been hampered by poor MSC graft survival, jeopardizing cellular and molecular bases for neural repair in vivo. We have devised an adult human bone marrow MSC (hMSC) delivery formula by investigating molecular events involving hMSCs incorporated in a uniquely designed poly(lactic-co-glycolic) acid scaffold, a clinically safe polymer, following inflammatory exposures in a dorsal root ganglion organotypic coculture system. Also, in rat T9-T10 hemisection spinal cord injury (SCI), we demonstrated that the tailored scaffolding maintained hMSC stemness, engraftment, and led to robust motosensory improvement, neuropathic pain and tissue damage mitigation, and myelin preservation. The scaffolded nontransdifferentiated hMSCs exerted multimodal effects of neurotrophism, angiogenesis, neurogenesis, antiautoimmunity, and antiinflammation. Hindlimb locomotion was restored by reestablished integrity of submidbrain circuits of serotonergic reticulospinal innervation at lumbar levels, the propriospinal projection network, neuromuscular junction, and central pattern generator, providing a platform for investigating molecular events underlying the repair impact of nondifferentiated hMSCs. Our approach enabled investigation of recovery neurobiology components for injured adult mammalian spinal cord that are different from those involved in normal neural function. The uncovered neural circuits and their molecular and cellular targets offer a biological underpinning for development of clinical rehabilitation therapies to treat disabilities and complications of SCI.

Keywords: PLGA; locomotion; mesenchymal stromal stem cell; recovery neurobiology; spinal cord injury.

Fig. S1.

hMSC quality control. (A and B) Immunostaining of MSC markers CD105 (A) and CD90 (B) for passage 12 (P12) hMSCs. (C) Oil Red O staining of adipogenic differentiation of P6–12 hMSCs (n = 6 per group; *, differentiation vs. control, P < 0.05, Student’s t test; ^, P6 vs. P9–12 and #, P9 vs. P11 and 12; P < 0.05, one-way ANOVA with Tukey’s post hoc test). (D) Real-time PCR showed mean expression levels of adiponectin (an adipogenic marker) in P8 and P11 hMSCs cultured in adipogenic or control medium (⁺, P8 vs. P11; statistical method per C). The real-time PCR finding was corroborated by Oil Red O staining of P12 and P6 hMSC-derived adipogenic cells shown in E and F, respectively. Alizarin Red S staining confirmed osteogenic capability of hMSCs. Whereas P9 hMSCs cultured in control condition showed no reactivity (G), the cells had high osteogenic yield after differentiation medium induction (H and I). Densitometric analysis of Alizarin Red S staining of P9 hMSCs cultured in osteogenic or control medium. Real-time PCR assay detected a significantly higher group mean level of ALP expression, an osteogenic marker, in P8 and P11 hMSCs cultured in osteogenic medium than in control culture and in P8 than in P11 hMSCs (J; statistical methods per C). Alcian Blue staining showed P9 cells with chondrogenic differentiation (K), and one of the intact chondrogenic pellets derived from hMSCs (L, a) that was cut (L, b) into 50-µm sections (L, c).

Fig. 1.

Multimodal effects of hMSCs in vitro. Compared with saline control, LPS or IFN exposure significantly augmented IDO expression (red) in both (A) nonscaffolded hMSCs and (B) PLGA-scaffolded hMSCs, as shown by human nuclei (hN) immunostaining (green; P < 0.05: *, control vs. LPS; ^#, control vs. IFN-γ; n = 5; one-way ANOVA with Tukey’s post hoc test). PLGA scaffolds showed yellow autofluorescence under dual channels. (C–E) DRG in organotypic coculture grew neurites (GAP43+, green) that track toward nearby hMSCs (CD90+, red; arrows), compared with (F) a more radial neurite pattern in DRG cultured with scaffold only (G). There were significantly more angular neurite paths in DRG and scaffolded hMSC cocultures (59.25 ± 2.9°) relative to the DRG and scaffold-alone group (48.15 ± 4.1°; P = 0.026, Student’s t test). Images showed different lengths of neurite outgrowth at the (H) distal and (I) proximal sites of axotomized DRG cocultured with PLGA-scaffolded hMSCs or scaffold alone, respectively. (J, Upper) The scaffolded hMSC and DRG cocultures had significant increases in mean total lengths when 3–20 neurites per DRG were averaged (*P = 0.032, n = 6, Mann–Whitney) and also (J, Lower) significantly increased the maximum absolute length of DRG neurite outgrow when 3–15 neurites per DRG were assessed (*P = 0.026, n = 6, Mann–Whitney). (K) Relative CNTF mRNA expression in scaffolded hMSCs was significantly elevated, as was (L) secretion of human BDNF (P < 0.05, one-way ANOVA) in the scaffolded hMSC + DRG coculture system.

Fig. S2.

Characterization of scaffolded hMSCs. (A) H&E staining of hMSCs seeded in PLGA scaffold. The PLGA polymer scaffold has a porous, soft, and smooth texture (Materials and Methods). (B) Immunostaining revealed extensive BDNF expression in hMSCs seeded in PLGA scaffold (red, BDNF; blue, DAPI staining of cell nuclei; green, PLGA autofluorescence). (C) Immunocytochemical stain for CD90 showed a high level of hMSC engraftment in the tailored PLGA scaffold (red, CD90; blue, DAPI; green, PLGA autofluorescence).

Fig. S3.

Antiinflammatory mechanisms of hMSCs investigated in an organotypic coculture system of axotomized DRG explants. (A–D) Generation of LPS-induced inflammation in an original axotomized DRG explant model. (A) Adult female rat DRG were explanted and embedded in Matrigel and treated for 2 h with LPS at various concentrations. Relative mRNA expression of TNFα, an inflammatory marker, was quantified with real-time PCR. A dose-dependent effect of LPS on DRG expression of TNFα plateaued at LPS doses ≥1,000 ng/mL. (B–D) Rat DRG explants were treated for 0.5, 6, 12, or 24 h with 2 ng/mL LPS. Relative mRNA expression levels of TNFα (B), IL-1 (C), and IL-6 (D) characteristic inflammatory cytokines were quantified by real-time PCR. (E–G) Organotypic DRG and scaffolded hMSCs cocultures were treated with 10 ng/mL LPS for 2 h. Fold difference in rat inflammatory cytokine mRNA levels for (E) TNFα, (F) IL-1, and (G) IL-6 are expressed relative to those of control DRG cultured with PLGA scaffolds coated with hMSC-conditioned medium only. Coculture with scaffolded hMSCs significantly suppressed expression of proinflammatory cytokines by axotomized DRG after LPS exposure (*P < 0.05, n = 6 per group; one-way ANOVA with Tukey’s post hoc test).

Fig. 2.

Treatment designs and behavioral outcomes. Schematic presentations of (A) the in vitro hMSC seeding process for a PLGA scaffold and (B) design of the T9–T10 midline hemisection injury followed by implant insertion. Compared with three control groups, treatment with PLGA-scaffolded hMSCs significantly improved overall coordinated motor function as determined by (C) group mean BBB locomotion score of the hindlimb ipsilateral to injury and (D) inclined plane angle. Implantation of scaffolded hMSCs also significantly reduced occurrence of abnormal spinal reflexes in response to (E) pressure and (G) contact-triggered righting. Both (F) brief nociceptive pinch to the toe pads and (H) sensory tests at T9–T10 with standard 2- and 10-g Semmes-Weinstein filaments showed markedly higher hypersensitive responses in the controls relative to the scaffolded hMSC-treated rats (*, treated vs. lesion control, P < 0.05, Fisher’s exact test). Data points (n = 7) represent average ± SEM or percent with normal (E–G) or abnormal (H) responses of each group, analyzed with repeated-measures ANOVA that showed an overall significant effect of treatment (P < 0.05). Symbols indicate that means are significantly different from those of the lesion-only (*), scaffold-only (#), and hMSCs-alone (^) control groups at the specified times after injury (Tukey’s post hoc procedure or Student’s t test).

Fig. 3.

Histopathological analysis. Solvent blue- and hematoxylin-stained serial transverse spinal cord sections showed (A) that spinal cords with implanted scaffolded hMSC had more spared tissue around the lesion site than hMSC-alone and lesion-only controls and complete degradation of the PLGA scaffolds at ≥6 wk after SCI. Quantitative assessment of representative spinal cords (n = 4 per group) showed that relative to the controls, (B) scaffolded hMSC significantly reduced mean lesion volume and (C) increased white matter sparing in the spinal cord sections ±2 mm from the lesion epicenter. (D) Scaffolded hMSC implantation also preserved ventral horn motor neurons (Left) in spinal cord tissue caudal to the epicenter. Asterisks indicate a significant difference from the control group (P < 0.05, one-way ANOVA or repeated-measures ANOVA with Tukey’s post hoc test).

Fig. 4.

Analysis of fate and multimodal effects of scaffolded hMSCs in vivo. (A) Immunostaining for CD90 and human heat shock protein 27 (hHSP27) showed long-term survival (≥6 wk postinjury) only of scaffolded hMSCs (arrowed cells in Insets for the framed area). The engrafted hMSCs were mostly CD90+ and primarily concentrated in the lesion epicenter, with sharply reduced presence in the adjacent parenchyma. (B) Costaining for hHSP27 demonstrated cytoplasmic BDNF in the engrafted hMSCs (Left); relative to the controls, mean expression level increased nearly fourfold in the scaffolded hMSC-treated group (Right: ^, comparing with hMSC alone or *, lesion only; n = 5 per group; P < 0.05). (C) Costaining for CD90 showed IL-10 expression in the engrafted hMSC cytoplasm (Left); expression level of IL-10 was significantly higher in the scaffolded hMSC-treated spinal cords (^, comparing with hMSC-only group and *, to lesion-only group; ⁺, hMSC only vs. lesion only; n = 5 per group; P < 0.05, one-way ANOVA with Tukey’s post hoc test). (D) Confocal analysis of the scaffolded hMSC-treated spinal cord tissue detected no discernible IR to collagen I, II, and IV or ALP (bone tissue marker), despite persistent presence of CD90+ signals, suggesting that no mesenchymal phenotypic differentiation of hMSCs occurred in the injured spinal cords ≥6 wk after transplantation. (E) Confocal z-stack imaging confirmed that IDO was expressed mainly in the cytoplasm of donor hMSCs (CD90+, green) in the subacutely injured spinal cord (i.e., 7–10 d postinjury). (F) The number of infiltrated CD3+ (red) T-cells was significantly decreased (G) in the white matter areas 3 mm rostral (Left) and caudal (Right) to the epicenter, and in the gray matter 3 mm caudal to the epicenter (G, Center). (H) Scaffolded hMSCs (CD90+, green) discernibly increased the number of macrophages manifesting M2 phenotype polarization (arginase 1 IR: green), whereas the number of activated microglia and macrophage (CD68+) was greatly reduced near the epicenter of the subacutely injured spinal cord, compared with lesion-only or hMSC-alone controls (n = 4 per group).

Fig. 5.

Investigation of neural repair effects of scaffolded hMSC engraftment. Transverse spinal cord sections, 3 mm caudal to the injury epicenters, were immunostained for (A) GFAP for astrogliosis; (B) nitrotyrosine (NT) for oxidative damage; (C) CD68 for activated microglia (and vascular-derived macrophages); (D) DCX for endogenous NSCs; (E) laminin for angiogenesis; and (F) human laminin (h-laminin) to assess if donor hMSCs produced these glycoproteins to promote neurite outgrowth. The scaffolded hMSC treatment significantly reduced (Right) IR of GFAP, NT (protein nitration), and CD68. The treatment significantly augmented endogenous neurogenesis (arrows and arrowheads denote DCX+ NSCs in D) and increased angiogenesis in chronically lesioned spinal cord, indicated by the blood vessel-resembling morphology of pan-laminin IR (arrowheads in E). Moreover, h-laminin secretion from hMSCs (CD90+; green) near the epicenter in the subacutely injured spinal cord was detected (F, i: h-laminin α2 chain; ii: h-laminin α5 chain). Last, h-laminin deposition persisted chronically to ≥6 wk after injury in the same region (F, iii: h-laminin α2 chain) and showed intimate interactions with 5-HT+ axonal fibers, suggesting its promotion of serotonergic reinnervation (F, iii). (Scale bar: A–D, 50 µm: E, 20 µm; F, 25 µm.) P < 0.05, one-way ANOVA with Tukey’s post hoc test: *, scaffolded hMSCs vs. lesion only; #, scaffolded hMSCs vs. hMSC alone, and ⁺, hMSC alone vs. lesion alone.

Fig. 6.

Neurobiological benefits resulting from the scaffolded hMSC treatment. (A) Tracing regimens: BDA (green; injected into motosensory cortex contralateral to hemisection) for tracing the CST on the lesioned side, whereas DiI, Fast Blue (FB), and Fluoro-Gold (FG) were injected ipsilaterally into cervical region muscles and intercostal and abdominal muscles (Materials and Methods) to trace both primary proprioceptive axons that synapse with the PSN interneurons and with ventral horn motor neurons. DiI and FG were also injected into hindlimb muscles for retrograde tracing of lumbar motor neurons and primary afferent fibers. (B) Schematic of a simplified PSN network consisting of cross-segmental 1° and 2° neurites that project bilaterally and bidirectionally. (C) BDA IR was detected in the ipsilateral CST rostral to the injury epicenter, but not at any levels caudal to the lesion site in both scaffolded hMSC-treated and lesion-only spinal cords, suggesting no CST regeneration. (D and E) Images of DiI-, FB-, or FG-traced cervical and thoracic ventral horn motor neurons, respectively, and the synapses in the surrounding regions that were costained by antibody against synaptophysin (Syn, green), a synaptic vesicle marker. Scaffolded hMSCs-treated spinal cords showed much higher Syn IR density in both cervical and thoracic cord regions, compared with lesion-only (or hMSCs-only) controls (F; P < 0.05, paired Student’s t test, n = 5 per group). (G, Left) Tissue slices selected from upper lumbar segment 5 mm caudal to the epicenter were immunostained for 5-HT (red) and vGluT1 (green). A representative z-stack confocal plane (20 × 0.1 μm optical sections) of the Rexed Lamina VIII area is shown (Inset) at 600× magnification, and presents typical details of the pericellular distribution of axon terminal vGluT1 and beaded varicosities of 5HT. (Scale bar: 5 µm.) (G, Right) Group average IR of vGluT1 (red) that marks primary proprioceptive terminals and 5HT (green) in lumbar Rexed Lamina VIII were significantly stronger in scaffolded hMSC-treated spinal cords compared with controls. (H) Coimmunostaining of STRO-1 (red), an hMSC marker and Cx43 (green), a gap junction protein, revealed significantly higher signal density of Cx43 in the host ventral cord region of sections immediately caudal to the scaffolded hMSC implant site, compared with the control groups (*, scaffolded hMSC vs. hMSC only and lesion only; #, hMSC only vs. lesion only; P < 0.05, n = 5 per group; one-way ANOVA with Tukey’s post hoc test). Cx43 was expressed mainly by host cells (i.e., STRO-1–negative cells). (I) Retrograde tracing with FG or DiI via T6–T8 intercostal nerve showed FG+ or DiI+ primary afferent fibers and (J) DRG neurons. Scaffolded hMSC treatment drastically increased densities and area of both tracers, relative to the lesion controls, and DRG neuron integrity (J). (K) Motor nerve endings are in close contact with α-bungarotoxin+ nicotinic receptors in the muscles, showing near normal morphology of NMJs in the quadriceps of scaffolded hMSC-treated rats.

Fig. S4.

Assessment of RST innervation in the spinal cord by vGluT2 immunostain. (A) RST, located in the dorsal aspect of the lateral funiculus (Insets) showed innervation along the side of the spinal cord ipsilateral to and above T9–T10 hemisection in the scaffolded hMSC implantation group, with immunocytochemistry detecting vGluT2 that is carried by all RST terminals at C8 segment (area details in B). By contrast, no vGlut2 immunoreactive axon terminals or presence of RST neurites were detected in similar areas of spinal cord segments below the SCI site (C: at the L2 level with detail in D), indicating no regeneration of RST axons across the epicenter following scaffolded hMSC treatment. (Scale bars: A and C, 200 µm; B and D, 30 µm.)