Early Immunomodulation by Intravenously Transplanted Mesenchymal Stem Cells Promotes Functional Recovery in Spinal Cord Injured Rats

Abstract

Although intravenous administration of mesenchymal stem cells (MSCs) can enhance functional recovery after spinal cord injury (SCI), the underlying mechanisms have to be elucidated. In this study, we explored the mechanisms for functional recovery in SCI rats after intravenous transplantation of MSCs derived from human umbilical cord blood. Sprague-Dawley rats were randomly assigned to receive either MSCs (1 × 10(6) cells/0.5 ml) or PBS into the tail vein immediately after SCI. They were then evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating scale weekly for 8 weeks and by somatosensory evoked potentials (SSEPs) 8 weeks after transplantation. MSC-treated rats showed a modest but significant improvement in BBB scores and latencies of SSEPs, compared with PBS controls. When human-specific Alu element was measured in the spinal cord, it was detected only 1 h after transplantation, suggesting transient engraftment of MSCs. Inflammatory cytokines were also determined using RT-PCR or Western blot in spinal cord extracts. In MSC-treated rats, the level of proinflammatory cytokine IL-1β was decreased, but that of anti-inflammatory cytokine IL-10 was increased. MSCs also immediately suppressed IL-6 at 1 h posttransplantation. However, the response of IL-6, which has an immunoregulatory role, was increased 1-3 days after transplantation. In addition, we quantified microglia/macrophage stained with Iba-1 around the damaged spinal cord using immunohistochemistry. A proportion of activated microglia and macrophages in total Iba-1(+) cells was significantly decreased in MSC-treated rats, compared with PBS controls. These results suggest that early immunomodulation by intravenously transplanted MSCs is a potential underlying mechanism for functional recovery after SCI.

Keywords: Functional recovery; Immunomodulation; Mesenchymal stem cells (MSCs); Spinal cord injury; Transplantation.

Figures

Figure 1

Characterization of MSCs. (A) MSCs were isolated from hUCB and cultured under LG-DMEM containing 10% FBS and 100 U/ml penicillin/streptomycin. Scale bar: 100 μm. (B) Spindle-shaped fibroblastic cells were characterized in high magnification. Scale bar: 100 μm. (C) Cells were labeled with FITC- and PE-conjugated antibodies and examined by flow cytometry. hUCB, human umbilical cord blood; MSCs, mesenchymal stem cells; LG-DMEM, low glucose Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 2

Locomotor and somatosensory assessment after intravenous transplantation of MSCs. (A) When locomotor performance was evaluated using the BBB locomotor rating scale, SCI rats treated with MSCs exhibited a gradual improvement over time. Namely, MSC-treated rats showed a modest but significant improvement in locomotor function 7–8 weeks (D49–D56) after transplantation, compared with PBS controls (*p < 0.05). (B) In addition, MSC-treated rats showed a representative finding of the shortened N1 latency and P1 latency of SSEPs compared to PBS controls. Values are mean ± SE. BBB, Basso-Beattie-Bresnahan; D, day; MSCs, mesenchymal stem cells; PBS, phosphate-buffered saline; SSEPs, somatosensory evoked potentials.

Figure 3

Genomic DNA PCR of human Alu element after intravenous transplantation of MSCs. (A) To determine the engraftment of intravenously injected MSCs in the spinal cord, expression of human Alu gene was examined at the indicated time points after MSC transplantation. The human-specific Alu was detected in injured spinal cord extractions only 1 h after transplantation but not detected thereafter, suggesting that MSCs disappeared after transient engraftment. (B) On the contrary, the human Alu gene was clearly detected in the lungs and spleen 1 h and 1 day after transplantation of MSCs, but not 7 days after transplantation. hUCB, human umbilical cord blood; MSCs, mesenchymal stem cells; PBS, phosphate-buffered saline; hAlu, human Alu; Cyclo, cyclophilin; M, marker. (+): positive control, the human hepatoma Hep3B cells.

Figure 4

Expression of inflammatory cytokines after intravenous transplantation of MSCs. (A) RT-PCR study showed an increase of anti-inflammatory cytokine IL-10 in MSCs-treated rats especially 1 day after transplantation. (B) In Western blot results, IL-10 was highly expressed in MSC-treated rats until at least 2 days after transplantation. In addition, proinflammatory cytokine IL-1β was modestly decreased after transplantation of MSCs. TNF-α seems to be downregulated 3 days after transplantation. MSCs also immediately suppressed proinflammatory cytokine IL-6 at 1 h after transplantation. However, the response of rat IL-6, which has an immunoregulatory role as well, was increased 1-3 days after intravenous administration of MSCs. MSCs, mesenchymal stem cells; PBS, phosphate-buffered saline; TNF, tumor necrosis factor; IL, interleukin; N, normal spinal cord.

Figure 5

Immunohistochemistry after intravenous transplantation of MSCs. (A, B) When we investigated the injured tissues in MSC-treated rats (A) and PBS controls (B) 8 weeks after transplantation, there was no significant difference in the mean area of cavitary lesion per section between them. Scale bars: 500 μm. (C) When we also performed immunostaining with human-specific HuNu and neural-lineage markers in animals treated with MSCs, only a few cells were suspected to be double-labeled with MBP at posttransplantation 4 weeks. Scale bar: 10 μm. (D–G) The sections of SCI rats treated with MSCs (D, E) and PBS (F, G) were costained for DAPI and Iba-1, which are highly expressed in microglia/macrophage. Scale bars: 10 μm. (H–K) The Iba-1+ cells were divided into two groups: ramified form representing resting microglia (H, I) and ameboid form representing activated microglia and macrophage (J, K). Scale bars: 10 μm. (L) When we compared the proportion of activated microglia/macrophage among total Iba-1+ cells between the groups 1 day after transplantation when the inflammatory cascades temporally peaked, a proportion of the activated microglia/macrophage was significantly suppressed in MSC-treated rats, compared with PBS-treated controls (*p < 0.05). MBP, myelin basic protein; Iba-1, ionized calcium binding adaptor molecule-1; DAPI, 4′,6-diamidino-2-phenylindole; MSCs, mesenchymal stem cells; PBS, phosphate-buffered saline.